Humanized antibodies

ABSTRACT

Humanized antibodies that bind ICAM-1 are provided. Antibodies include those selected from: SEQ ID NO:1 and 3 (HumA); SEQ ID NO:5 and 7 (HumB); SEQ ID NO:9 and 11 (HumC); SEQ ID NO:13 and 15 (HumD); SEQ ID NO:17 and 19 (HumE); SEQ ID NO:21 and 23 (HumF); SEQ ID NO:25 and 27 (HumG); SEQ ID NO:29 and 31 (HumH); and SEQ ID NO:33 and 35 (HumI). Subsequences of the humanized antibodies capable of binding an ICAM-1 epitope are also provided. Methods of inhibiting pathogen infection (e.g., HRV) of a cell employing humanized antibodies capable of binding an ICAM-1 epitope are further provided.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part and claims the benefitof priority of application Ser. No. 09/555,446, filed May 31, 2001,which is a 35 U.S.C. 371 of PCT application serial no. PCT/US98/25422,filed Nov. 30, 1998.

FIELD OF THE INVENTION

[0002] The invention relates to humanized antibody compositions andmethods of making and using humanized antibodies.

BACKGROUND

[0003] Monoclonal antibodies have become an important class oftherapeutic proteins. However, foreign immunoglobulins used in humanscan elicit an anti-globulin response which may interfere with therapy orcause allergic or immune complex hypersensitivity. To avoid thisproblem, a monoclonal antibody may be “humanized,” and this is typicallycarried out by CDR grafting.

[0004] CDR's, also called hypervariable regions, are present inimmunoglobulin light and heavy chains and are flanked by “framework”regions. CDR grafting was first described in Jones et al. ((1986) Nature321:522-525). In this and later publications, the CDRs of three mouseantibodies were grafted onto the variable domain frameworks of the humanimmunoglobulin NEW (V_(H)) and REI (V_(L)). The resulting humanizedantibodies had the same antigen specificity and a similar affinity asthe parental murine monoclonal antibody (mAb) (Jones et al. supra;Verhoeyen et al. (1988) Science 239:1534-1536; Riechmann et al. (1988)Nature 332:323-327; U.S. Pat. No. 5,225,539).

[0005] CDR grafting has been described by Queen and coworkers whoreported the humanization of four murine monoclonal antibodies (Queen etal. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033; Co et al. (1991)Proc. Natl. Acad. Sci. USA 88:2869-2873; Co et al. (1992) J.Immunol.148:1149-1154; and U.S. Pat. Nos. 5,585,089; 5,693,761; and5,693,762). Murine residues were inserted in the human framework inorder to maintain affinity and, in each case the original antigenspecificity was maintained. The affinities of the humanized antibodiesranged from 1/3 to 3 times of the parental unmodified murine antibodies.

SUMMARY

[0006] The invention provides humanized antibodies that bind ICAM-1. Inone embodiment, the antibody is selected from: SEQ ID NO:1 and 3 (HumA);SEQ ID NO:5 and 7 (HumB); SEQ ID NO:9 and 11 (HumC); SEQ ID NO:13 and 15(HumD); SEQ ID NO:17 and 19 (HumE); SEQ ID NO:21 and 23 (HumF); SEQ IDNO:25 and 27 (HumG); SEQ ID NO:29 and 31 (HumH); and SEQ ID NO:33 and 35(HumI). Subsequences of antibodies that bind ICAM-1 are provided, forexample, single chain, Fab, Fab′ and (Fab)₂ fragments. In particularaspects, the humanized antibody has greater affinity for ICAM-1 than theparental (non-human) antibody. Varaint and modified forms of antibodiesthat bind ICAM-1 are also provided, for example, antibodies selectedfrom SEQ ID NO: 1 and 3 (HumA); SEQ ID NO:5 and 7 (HumB); SEQ ID NO:9and 11 (HumC); SEQ ID NO:13 and 15 (HumD); SEQ ID NO:17 and 19 (HumE);SEQ ID NO:21 and 23 (HumF); SEQ ID NO:25 and 27 (HumG); SEQ ID NO:29 and31 (HumH); and SEQ ID NO:33 and 35 (HumI) having one or more amino acidsubstitutions, insertions or deletions.

[0007] The invention also provides humanized antibodies that bind ICAM-1and inhibit pathogen infection of cells expressing ICAM-1. Suchinvention antibodies include antibodies that provide greater protectionfrom pathogen infection than parental (non-human) antibody. Inparticular aspects, a humanized antibody has a protective efficacy atleast 2 times greater, 5 times greater, 10 times greater, 20 timesgreater, 30 times greater than the non-humanized antibody. In otheraspects, the pathogen is human rhinovirus (HRV), coxackie A virus,respiratory syncytial virus (RSV), or malaria.

[0008] The humanized antibodies of the invention include intactimmunoglobulin molecules, comprising 2 full-length heavy chains and 2full-length light chains, and subsequences that inhibit pathogeninfection. Particular subsequences include, for example, single chain,Fab, Fab′ or (Fab)₂ fragment.

[0009] The humanized antibodies of the invention include multispecificor multifunctional antibodies. In one aspect, such an antibody is formedby linking a humanized antibody to one or more identical or differentantibodies to form a multimer (e.g. using a linker). Antibody multimersinclude a homo- or hetero-dimer, trimer, tetramer or any other higherorder oligomer. Antibody multimers that include different antibodies arehuman, humanized or non-human. Multimeric forms include antibodyoligomers that form via a multimerization domain (e.g. a human aminoacid sequence) or a covalent bond. Antibody multimers that include amultimerization domain further include forms having a linker locatedbetween the multimerization domain and the antibody.

[0010] The invention additionally provides nucleic acid sequencesencoding humanized antibodies, subsequences and modified froms thereof(e.g., amin acid additions, deletions or substitutions). Nucleic acidsequences further include expression cassettes in which nucleic acidencoding humanized antibodies are operably linked to an expressioncontrol element. Vectors and cells (prokaryotic and eukaryotic) thatinclude the nucleic acids also are provided.

[0011] The invention further provides pharmaceutical compositionsincluding humanized antibodies, subsequences, multimers, variants andmodified forms, and nucleic acids encoding them, and a pharmaceuticallyacceptable carrier. In particular aspects, the pharmaceuticallyacceptable carrier is compatible with inhalation or nasal delivery to asubject.

[0012] The invention further provides methods of inhibiting pathogeninfection of a cell. In one embodiment, a method includes contacting apathogen or a cell with an amount of a humanized antibody, subsequence,multimer, variant or modified form sufficient to inhibit pathogeninfection of the cell. In one aspect, the cell expresses ICAM-1. Inanother aspect, the cell (e.g., epithelial cell) is present in asubject.

[0013] The invention also provides methods of inhibiting HRV infectionof a cell. In one embodiment, a method includes contacting HRV or a cellsusceptible to HRV infection with an amount of a humanized antibody,subsequence, multimer, variant or modified form effective to inhibit HRVinfection of the cell (e.g., epithelial cell). In one aspect, the cellis present in a subject. In another aspect, the cell is present in asubject having or at risk of having asthma. In yet another aspect, thesubject is a newborn or between the ages of 1 to 5, 5 to 10 or 10 to 18.In still another aspect, the antibody, subsequence, multimer, variant ormodified form binds to an antigen present on the surface of the cell(e.g., ICAM-1). In various additional aspects, the humanized antibody isadministered locally, via inhalation or intranasaly.

[0014] The invention also provides methods of inhibiting HRV infection,inhibiting HRV progression or treating HRV infection of a subject. Inone embodiment, a method includes administering to a subject having orat risk of having HRV infection an amount of a humanized antibody,subsequence, multimer, variant or modified form effective to inhibit HRVinfection, inhibit HRV progression or treat HRV infection of thesubject. In one aspect, the subject has or is at risk of having asthma.In another aspect, the subject is a newborn or between the ages of 1 to5, 5 to 10 or 10 to 18. In various additional aspects, the humanizedantibody is administered locally, via inhalation or intranasaly.

[0015] The invention additionally provides methods of decreasing orinhibiting one or more symptoms of the common cold in a subject. In oneembodiment, a method includes administering to a subject having a commoncold an amount of a humanized antibody, subsequence, multimer, variantor modified form effective to decrease or inhibit one or more symptomsof the common cold in the subject. In one aspect, the subject has or isat risk of having asthma. In another aspect, the subject is a newborn orbetween the ages of 1 to 5, 5 to 10 or 10 to 18. In various additionalaspects, the humanized antibody is administered locally, via inhalationor intranasaly.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 shows the amino acid sequence of murine 1A6 antibody andhuman consensus sequence of heavy chain subgroup III (Humiii) and lightchain kappa subgroup I. Asterisks denote amino acid differences betweenhuman and mouse sequence. CDR amino acids are in bold face.

[0017]FIG. 2 shows a molecular model of humanized 1A6 (Hum B). (A) Sideview of humB variable domains. Yellow: V_(H); green: V_(L); pale blue:CDRs; red: the six high risk “Vernier zone” residues and the V_(H)60-64. (B) Top view of humB variable domains. Yellow: V_(H); green:V_(L); pale blue: CDRs; red: the six high risk “Vernier zone” residuesand the V_(H)60-64. (C) V_(L)49 and its surrounding residues. Magenta:ICAM-1; red: V_(H) and V_(L) residues; blue: residues at V_(H)-V_(L)interphase; purple: W102 in V_(H).

[0018]FIG. 3 shows the amino acid sequence of murine 1A6 antibody,humanized 1A6 (HumB) and human consensus sequences of heavy chainsubgroup III (Humiii) and light chain kappa subgroup I. Asterisks andbold face amino acids are as previously indicated.

[0019]FIG. 4 shows the cDNA sequence of humanized scFV3 (HumA) antibody.Restriction sites are indicated by underlining.

[0020]FIG. 5 shows protection from HRV15 infection with mouse 1A6 scFvantibody and humanized 1A6 scFv antibody HumA, HumB, HumC, HumD, HumF,HumH and HumI.

DETAILED DESCRIPTION

[0021] The present invention is based, at least in part, upon producinghumanized antibodies. More particularly, complementarity determiningregion (CDR) from a non-human antibody are grafted into a humanframework region. Following grafting, one or more amino acids of theantibody are mutated to human sequences. For example, mutating a murineamino acid to a human amino acid in a framework region or CDR of thegrafted antibody can produce a humanized antibody having increasedantigen binding affinity relative to the non-human or grafted antibody.Humanized antibodies are not immunogenic or are less immunogenic thannon-human antibodies when administered to human subjects. Therefore,humanized antibodies are useful in a variety of therapeutic anddiagnostic applications. For example, as exemplified herein, a humanizedantibody of the invention protects cells from HRV infection, a virusthat can cause the common cold, and other associated disorders (e.g.otitis media, bronchitis, sinusitis etc.).

[0022] Thus, in accordance with the invention, there are providedhumanized antibodies. In one embodiment, a humanized antibody binds toICAM-1. In one aspect, a humanized antibody that binds ICAM-1 protectsagainst pathogen infection of cells expressing ICAM-1. In other aspects,a humanized antibody is selected from any of SEQ ID NO: 1 and 3 (HumA);SEQ ID NO:5 and 7 (HumB); SEQ ID NO:9 and 11 (HumC); SEQ ID NO:13 and 15(HumD); SEQ ID NO:17 and 19 (HumE); SEQ ID NO:21 and 23 (HumF); SEQ IDNO:25 and 27 (HumG); SEQ ID NO:29 and 31 (HumH); and SEQ ID NO:33 and 35(HumI). In another embodiment, a humanized antibody has a greater orless affinity for the antigen than the donor non-human antibody. Invarious aspects, affinities range from greater or less affinity for theantigen than either the donor or recombinant antibody. In particularaspects, humanized antibody has an antigen binding affinity 4-fold,5-fold, 5- to 8-fold, 5- to 10-fold, 8- to 15-fold, 10- to 20-fold, 20-to 100-fold or greater than the parental antibody.

[0023] Human antibody sequence regions can be used for producinghumanized antibodies of the invention. For example, a “consensussequence,” an antibody sequence having the most frequently occurringamino acid residues at particular positions in an antibody or anantibody region, may be used. As an example, human variable regiondomain sequences are described in Kabat (Sequences of Proteins ofImmunological Interest. 4^(th) Ed.US Department of Health and HumanServices. Public Health Service (1987)). Sequences that are completelydetermined in the framework regions, 1-23, 35-49, and 57-88 in the lightchains, and in the framework regions, 1-30, 36-49, and 66-94, in theheavy chains, are included in the survey. For the fourth frameworkregion, 98-107 in the light chain and 103-113 in the heavy chain,residues that can be derived from the known J-minigene segments aresurveyed.

[0024] At the end of the survey, the most frequently occurring residueat a given position is chosen as the residue in the consensus sequence.Consensus sequences may therefore be identified by surveying amino acidresidues at each position of a plurality of antibodies; the mostfrequently occurring amino acid at a given position in the region ofinterest is a part of the consensus. In many instances, more than oneresidue will be found at high frequency at a given position. In suchcases, if the amino acid that occurs at least one-fourth as frequentlyas the most frequently occurring the amino acid residue is considered apart of the consensus sequence.

[0025] The published consensus sequence of human V_(H) subgroup III,based on a survey of 22 known human V_(H) III sequences, and thepublished consensus sequence of human V_(L) kappa-chain subgroup I,based on a survey of 30 known human kappa I sequences may be used forhuman antibody (Padlan (1994) Mol. Immunol. 31:169-217; Padlan (1991)Mol. Immunol. 28:489-498). These human consensus sequences werepreviously used to humanize two antibodies (Carter et al. (1992) Proc.Natl. Acad. Sci. USA 89:4285-4289; Presta et al. (1993) J. Immunol. 151:2623-2632). These human V_(H) subgroup III and V_(L)-kappa subgroup Iconsensus sequences are selected as frameworks, respectively, tohumanize mAb1A6 as described in Example 1. Thus, consensus sequencesknown in the art, as exemplified for human V_(H) subgroup III orV_(L)-kappa subgroup I, can be chosen as acceptor frameworks forproducing humanized antibody in accordance with the invention.

[0026] Any mouse, rat, guinea pig, goat, non-human primate (e.g., ape,chimpanzee, macaque, orangutan, etc.) or other animal antibody may beused as a CDR donor for producing humanized antibody. Murine antibodiessecreted by hybridoma cell lines can also be used. Donor CDRs areselected based upon the antigen to which the antibody binds. Thus, donorCDRs include sequences from antibodies that bind to pathogens, such asbacteria, viruses, protozoa and other microorganisms. Donor CDRs alsoinclude antibodies that bind to molecules to which the pathogens bind,for example, cell surface proteins (e.g., adhesion proteins, receptorproteins, immune-recognition/modulation proteins such as HLA, tumorassociated antigens, etc.). In one particular example, the donorantibody is a mouse monoclonal antibody 1A6 (mAb1A6), which specificallybinds to ICAM-1.

[0027] “Complementarity determining regions” or “CDRs” are among thesequences that can be grafted into framework sequences. CDRs refer tosequence regions that confer antibody specificity and affinity. CDRs arealso generally known as supervariable regions or hypervariable loops.CDR regions of antibodies have been mapped and are defined as in Kabat(Sequences of Proteins of Immunological Interest. 4^(th) Ed.USDepartment of Health and Human Services. Public Health Service (1987))and Chothia and Lesk ((1987) J. Mol. Biol. 186:651-663)). In particular,for heavy chain, CDR1 is defined as H26-H35, CDR2 is H50-65 and CDR3 isH95-H102; for light chain, CDR 1 is L24-L34, CDR2 is L50-L56 and CDR3 isL89-L97. The amino acids are numbered according to the scheme describedin Kabat (Sequences of Proteins of Immunological Interest. 4^(th) Ed.USDepartment of Health and Human Services. Public Health Service (1987)).Variable region domains typically comprise the amino-terminalapproximately 105-115 amino acids a of a naturally-occurringimmunoglobulin chain (e.g., amino acids 1-110). Variable domains shorteror longer than these exemplary sequence lengths may also be used.

[0028] Thus, the invention provides humanized antibodies, methods ofmaking the antibodies and methods of using the antibodies, includingtherapeutic and diagnostic methods. In one embodiment, a humanizedantibody has increased affinity for the antigen relative tonon-humanized antibody (e.g., less than 1.18×10⁻⁶ M in K_(D) againstICAM-1, less than 1×10⁻⁷ M in K_(D), less than 5×10⁻⁷ M in K_(D), lessthan 1×10⁻⁸ M in K_(D), less than 5×10⁻⁸ M in K_(D)). In variousaspects, a humanized antibody comprises an amino acid sequence set forthin any of SEQ ID NO:1 and 3 (HumA); SEQ ID NO:5 and 7 (HumB); SEQ IDNO:9 and 11 (HumC); SEQ ID NO:13 and 15 (HumD); SEQ ID NO:17 and 19(HumE); SEQ ID NO:21 and 23 (HumF); SEQ ID NO:25 and 27 (HumG); SEQ IDNO:29 and 31 (HumH); and SEQ ID NO:33 and 35 (HumI); and antigen bindingsubsequences thereof. In various additional aspects, an antibodysubsequence comprises Fab, Fab′, (Fab′)₂, Fv, and single chain antibody(SCA), e.g., scFv fragments.

[0029] The humanized antibodies of the invention also include antibodymultimers. In various aspects, a multimer comprises a dimer, trimer,tetramer or other higher order oligomer. In other aspects, multimerscomprise combinations of the same antibodies (homo-oligomers) anddifferent antibodies (hetero-oligomers), the different antibodies beinghuman, humanized or non-human.

[0030] The terms “protein,” “polypeptide” and “peptide” are usedinterchangeably herein to refer to two or more covalently linked aminoacids, also referred to as “residues,” through an amide bond orequivalent. Polypeptides are of unlimited length and may be comprised ofL- or D-amino acids as well as mixtures thereof. Amino acids may belinked by non-natural and non-amide chemical bonds including, forexample, those formed with glutaraldehyde, N-hydoxysuccinimide esters,bifunctional maleimides, or N,N′-dicyclohexylcarbodiimide (DCC).Non-amide bonds include, for example, ketomethylene, aminomethylene,olefin, ether, thioether and the like (see, e.g., Spatola (1983) inChemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol.7, pp 267-357, “Peptide and Backbone Modifications,” Marcel Decker,N.Y.). Polypeptides may have one or more cyclic structures such as anend-to-end amide bond between the amino and carboxy-terminus of themolecule or intra- or inter-molecular disulfide bond. Polypeptides maybe modified in vitro or in vivo, e.g., post-translationally modified toinclude, for example, sugar residues, phosphate groups, ubiquitin, fattyacids or lipids. Polypeptides further include amino acid structural andfunctional analogues, for example, peptidomimetics having synthetic ornon-natural amino acids or amino acid analogues.

[0031] The term “antibody” refers to a protein that binds to othermolecules (antigens) via heavy and light chain variable domains, V_(H)and V_(L), respectively. Antibodies include IgG, IgD, IgA, IgM and IgE.The antibodies may be intact immunoglobulin molecules, two full lengthheavy chains linked by disulfide bonds to two full length light chains,as well as subsequences (i.e. fragments) of immunoglobulin molecules,with our without constant region, that bind to an epitope of an antigen,or subsequences thereof (i.e. fragments) of immunoglobulin molecules,with or without constant region, that bind to an epitope of an antigen.Antibodies may comprise full length heavy and light chain variabledomains, V_(H) and V_(L), individually or in any combination. Forexample, each of SEQ ID NO:1 and 3 (HumA); SEQ ID NO:5 and 7 (HumB); SEQID NO:9 and 11 (HumC); SEQ ID NO:13 and 15 (HumD); SEQ ID NO:17 and 19(HumE); SEQ ID NO:21 and 23 (HumF); SEQ ID NO:25 and 27 (HumG); SEQ IDNO:29 and 31 (HumH); and SEQ ID NO:33 and 35 (HumI) are includedindividually and in any combination.

[0032] Polypeptide sequences can be made using recombinant DNAtechnology of polypeptide encoding nucleic acids via cell expression orin vitro translation, or chemical synthesis of polypeptide chains usingmethods known in the art. Antibodies according to the invention,including humanized sequences and subsequences can be expressed fromrecombinantly produced antibody-encoding nucleic acid (see, e.g., Harlowand Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1989; Harlow and Lane, Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, 1999; Fitzgerald et al., J. A. C.S. 117:11075 (1995); Gram et al., Proc. Natl. Acad. Sci. USA 89:3576-80(1992)). For example, as described in Example 3, cDNA encoding humanizedantibody sequences can be expressed in bacteria in order to produceinvention antibodies. Antibodies may also be produced by expressingencoding nucleic acids in mammalian, insect, and plant cells.Polypeptide sequences including antibodies can also be produced by achemical synthesizer (see, e.g., Applied Biosystems, Foster City,Calif.).

[0033] As used herein, the term “subsequence” or “fragment” means aportion of the full length molecule. For example, a subsequence of anantibody is one or more amino acid less in length than full lengthpolypeptide (e.g. one or more internal or terminal amino acid deletionsfrom either amino or carboxy-termini). Subsequences therefore can be anylength up to the full length molecule.

[0034] Specific examples of antibody subsequences include, for example,Fab, Fab′, (Fab′)₂, Fv, or single chain antibody (SCA) fragment (e.g.,scFv). Subsequences include portions which retain at least part of thefunction or activity of full length sequence. For example, an antibodysubsequence will retain the ability to selectively bind to an antigeneven though the binding affinity of the subsequence may be greater orless than the binding affinity of the full length antibody. Subsequencescan comprise a portion of any of the invention humanized sequences, forexample, any of SEQ ID NO: 1 and 3 (HumA); SEQ ID NO:5 and 7 (HumB); SEQID NO:9 and 11 (HumC); SEQ ID NO:13 and 15 (HumD); SEQ ID NO:17 and 19(HumE); SEQ ID NO:21 and 23 (HumF); SEQ ID NO:25 and 27 (HumG); SEQ IDNO:29 and 31 (HumH); and SEQ ID NO:33 and 35 (HumI).

[0035] Pepsin or papain digestion of whole antibodies can be used togenerate antibody fragments. In particular, an Fab fragment consists ofa monovalent antigen-binding fragment of an antibody molecule, and canbe produced by digestion of a whole antibody molecule with the enzymepapain, to yield a fragment consisting of an intact light chain and aportion of a heavy chain. An (Fab′)₂ fragment of an antibody can beobtained by treating a whole antibody molecule with the enzyme pepsin,without subsequent reduction. An Fab′ fragment of an antibody moleculecan be obtained from (Fab′)₂ by reduction with a thiol reducing agent,which yields a molecule consisting of an intact light chain and aportion of a heavy chain. Two Fab′ fragments are obtained per antibodymolecule treated in this manner.

[0036] An Fv fragment is a fragment containing the variable region of alight chain V_(L) and the variable region of a heavy chain V_(H)expressed as two chains. The association may be non-covalent or may becovalent, such as a chemical cross-linking agent or an intermoleculardisulfide bond (Inbar et al., (1972) Proc. Natl. Acad Sci. USA 69:2659;Sandhu (1992) Crit. Rev. Biotech. 12:437).

[0037] A single chain antibody (“SCA”) is a genetically engineered orenzymatically digested antibody containing the variable region of alight chain V_(L) and the variable region of a heavy chain, optionallylinked by a flexible linker, such as a polypeptide sequence, in eitherV_(L)-linker-V_(H) orientation or in V_(H)-linker-V_(L) orientation.Alternatively, a single chain Fv fragment can be produced by linking twovariable domains via a disulfide linkage between two cysteine residues.Methods for producing scFv antibodies are described, for example, byWhitlow et al., (1991) In: Methods: A Companion to Methods in Enzymology2:97; U.S. Pat. No. 4,946,778; and Pack et al., (1993) Bio/Technology11:1271.

[0038] Other methods of producing antibody subsequences, such asseparation of heavy chains to form monovalent light-heavy chainfragments, further cleavage of fragments, or other enzymatic, chemical,or genetic techniques may also be used, provided that the subsequencesbind to the antigen to which the intact antibody binds.

[0039] As used herein, the term “bind” or “binding” means that thecompositions referred to have affinity for each other. “Specificbinding” is where the binding is selective between two molecules. Aparticular example of specific binding is that which occurs between anantibody and an antigen. Typically, specific binding can bedistinguished from non-specific when the dissociation constant (K_(D))is less than about 1×10⁻⁵ M or less than about 1×10⁻⁶ M or 1×10⁻⁷ M.Specific binding can be detected, for example, by ELISA,immunoprecipitation, coprecipitation, with or without chemicalcrosslinking, two-hybrid assays and the like. Appropriate controls canbe used to distinguish between “specific” and “non-specific” binding.

[0040] Invention antibodies, including full length antibodies,subsequences (e.g., single chain forms) may be present as dimer,trimers, tetramers, pentamers, hexamers or any other higher orderoligomer that retains at least a part of antigen binding activity ofmonomer. Multimers can comprise heteromeric or homomeric combinations offull length antibody, subsequences, unmodified or modified as set forthherein and known in the art. Antibody multimers are useful forincreasing antigen avidity in comparison to monomer due to the multimerhaving multiple antigen binding sites. Antibody multimers are alsouseful for producing oligomeric (e.g., dimer, trimer, tertamer, etc.)combinations of different antibodies thereby producing compositions ofantibodies that are multifunctional (e.g., bifunctional, trifunctional,tetrafunctional, etc.).

[0041] The term “multifunctional” means that the composition referred tohas two or more activities or functions (e.g., antigen binding, enzymeactivity, ligand or receptor binding, toxin, etc.). For example, anantibody that binds to a particular antigen which also has an attachedpolypeptide with enzyme activity (e.g., luciferase, acetyltransferase,galactosidase, peroxidase, etc.) is one particular example of amultifunctional antibody.

[0042] Multifunctional antibodies further include multispecific (e.g.,bispecific, trispecific, tetraspecific, etc.) forms. The term“multispecific” means an antibody that binds to two or more differentantigenic epitopes. The term “multispecific” means that the antibodycontains two or more variable region sequences that bind to differentepitopes. The different epitopes may be present on the same antigen ordifferent antigens. For example, a multispecific antibody oligomercomprises a mixture of two or more antibodies each having differentepitope binding specificity and which form a multimer. Multispecificantibodies may be comprised of individual antigen binding polypeptideseach of which have distinct variable domains. For example, one of theantibodies may have two variable domains each of which recognizes adifferent epitope.

[0043] Candidate functions for multifunctional antibodies other thanantigen binding and in addition to enzyme activity include, for example,detectable moieties such as radioisotopes and amino acid sequences(e.g., ³⁵S, ¹³¹I, T7, immunoglobulin or polyhistidine tags, toxins(e.g., ricin, cholera, pertussis), cell surface proteins such asreceptors, ligands (substrates, agonists and antagonists), adhesionproteins (e.g., streptavidin, avidin, lectins), growth factors,differentiative factors and chemotactic factors.

[0044] Multifunctional humanized antibodies can be produced throughchemical crosslinking of the selected molecules (which have beenproduced by synthetic means or by expression of nucleic acid that encodethe polypeptides) or through recombinant DNA technology combined with invitro, or cellular expression of the polypeptide, and subsequentoligomerization. Multispecific antibodies can be similarly producedthrough recombinant technology and expression, fusion of hybridomas thatproduce antibodies with different epitopic specificities, or expressionof multiple nucleic acid encoding antibody variable chains withdifferent epitopic specificities in a single cell.

[0045] Antibodies may be either joined directly or indirectly throughcovalent or non-covalent binding, e.g. via a multimerization domain, toproduce multimers. A “multimerization domain” mediates non-covalentprotein-protein interactions. Specific examples include coiled-coil(e.g., leucine zipper structures) and alpha-helical protein sequences.Sequences that mediate protein-protein binding via Van der Waals'forces, hydrogen bonding or charge-charge bonds are also contemplated asmultimerization domains. Additional examples includebasic-helix-loop-helix domains and other protein sequences that mediateheteromeric or homomeric protein-protein interactions among nucleic acidbinding proteins (e.g., DNA binding transcription factors, such asTAFs). One specific example of a multimerization domain is p53 residues319 to 360 which mediate tetramer formation. Another example is humanplatelet factor 4, which self-assembles into tetramers. Yet anotherexample is extracellular protein TSP4, a member of the thrombospondinfamily, which can form pentamers. Additional specific examples are theleucine zippers of jun, fos, and yeast protein GCN4.

[0046] Humanized antibodies may be directly linked to each other via achemical cross linking agent or can be connected via a linker sequence(e.g., a peptide sequence) to form multimers. As used herein, “linker”or “spacer” refers to a molecule or group of molecules that connects twoor more molecules to each other. A flexible linker allows rotation ofthe two molecules linked to each other to the extent that the moleculesdo not block each others function. For example, a linker such as anamino acid sequence attached to a humanized antibody which is itselfattached to a multimerization domain, allows the antibody to bind toantigen without significant steric interference from the multimers ofthe oligomer. Non-peptide linkers include chemical cross linking agentsand polyethylene glycol.

[0047] One specific example of a peptide linker is an immunoglobulinhinge sequence. Additional specific examples are polylyisne,ployglutamic acid and mixtures of randomized amino acid sequences.Linker amino acid sequences may be fully human, humanized or non-humanamino acid sequences, unmodified or modified as set forth herein. Theinvention therefore further provides humanized antibodies that include alinker sequence. Linker sequences include, for example, sequences fromabout 2 to 10, 10 to 20, 10 to 30, 25 to 50, 30 to 60 and 50 to 75 aminoacids in length.

[0048] Antibodies also include modified forms such as sequences havingone or more amino acid substitutions, additions or deletions, providedthe modification does not destroy function, e.g., does not destroyantigen binding activity; the antibody retains, at least in part,antigen binding activity. For example, a modified humanized antibodywill retain, at least in part, affinity for the antigen to whichunmodified antibody binds. The term “modification” therefore denotes analteration of a molecule that does not destroy an activity of themodified molecule.

[0049] Modifications therefore include, for example, amino acidadditions, insertions, deletions and substitutions. An example of anaddition is where one or more amino acids are added to the N- orC-terminal end of a humanized antibody. An example of an insertion iswhere an amino acid is inserted into the sequence. An example of adeletion is where one or more amino acids are deleted from the N- orC-terminal end, or internally within the sequence.

[0050] The invention therefore also provides modified forms of thehumanized antibodies, including one or more amino acid additions,insertions, deletions and substitutions. In one embodiment, a humanizedantibody has one or more amino acid substitutions of a sequence setforth in SEQ ID NO: 1 and 3 (HumA); SEQ ID NO:5 and 7 (HumB); SEQ IDNO:9 and 11 (HumC); SEQ ID NO:13 and 15 (HumD); SEQ ID NO:17 and 19(HumE); SEQ ID NO:21 and 23 (HumF); SEQ ID NO:25 and 27 (HumG); SEQ IDNO:29 and 31 (HumH); and SEQ ID NO:33 and 35 (HumI), provided that thesubstituted antibody is capable of antigen binding. In a particularaspect, one or more of the amino acid substitutions are conservativeamino acid substitutions. In another aspect, the substitution comprises1-3, 3-5 or 5-10 amino acids. In yet another aspect, the substitution iswith a human amino acid.

[0051] Exemplary amino acid substitutions include conservative aminoacid substitutions. The term “conservative substitution” means thereplacement of one amino acid by a biologically or chemically similarresidue. Biologically similar means that the substitution is compatiblewith biological activity, e.g., for a humanized antibody, antigenbinding. Particular examples of conservative substitutions include thesubstitution of one hydrophobic residue, such as isoleucine, valine,leucine or methionine for another, or the substitution of one polarresidue for another, such as the substitution of arginine for lysine,glutamic for aspartic acids, or glutamine for asparagine, serine forthreonine, and the like.

[0052] Modifications also include derivatized sequences, for example,amino acids in which free amino groups form amine hydrochlorides,p-toluene sulfonyl groups, cabrobenzoxy groups; the free carboxy groupsfrom salts, methyl and ethyl esters; free hydroxl groups that formO-acyl or O-alkyl derivatives, as well as naturally occurring amino acidderivatives, for example, 4-hydroxyproline, for proline, 5-hydroxylysinefor lysine, homoserine for serine, ornithine for lysine, etc. Alsoincluded are modifications that confer covalent bonding, for example, adisulfide linkage between two cysteine residues thereby producing acyclic polypeptide. Modifications can be produced using any of a varietyof methods well known in the art (e.g., PCR based sited-directed,deletion and insertion mutagenesis, chemical modification andmutagenesis, cross-linking, etc.).

[0053] Modifications also include addition of functional entities suchas tags (e.g., polyhistidine, T7, immunoglubulin, etc.), gold particles,covalently or non-covalently attached to the humanized antibodies orsubsequences or multimers. Thus, the invention provides modifiedhumanized antibodies having one or more activities (e.g., retain atleast part of the antigen binding activity) of unmodified parentantibody. Modifications include radioactive or alternativelynon-radioactive detectable labels attached to or incorporated into themolecule.

[0054] The term “identical” or “identity” means that two or morereferenced entities are the same. Thus, where two nucleic acid sequencesare identical, they have the same sequence. “Areas of identity” meansthat a portion of two or more referenced entities are the same. Thus,where two nucleic acid sequences are identical over one or more parts oftheir sequence, they share identity in these areas. The term“substantial identity” means that the identity is structurally orfunctionally significant. That is, the identity is such that themolecules are structurally identical or perform the same function (e.g.,biological function) even though the molecules differ. Due to variationin the amount of sequence conservation between structurally andfunctionally related proteins, the amount of sequence identity forsubstantial identity will depend upon the type of region/domain and itsfunction. For nucleic acid sequences, 50% sequence homology and abovemay constitute substantial homology. Substantial homology for proteinscan be significantly less, for example, as little as 30% sequencehomology, but typically is more, e.g., 50%, 60%, 75%, 85% or more.

[0055] The extent of identity between two sequences can be ascertainedusing various computer programs and mathematical algorithms known in theart. Such algorithms that calculate percent sequence identity (homology)generally account for sequence gaps and mismatches over the comparisonregion. For example, a BLAST (e.g., BLAST 2.0) search algorithm (see,e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-10, publiclyavailable through NCBI at http:/www.ncbi.nlm.nih.gov) has exemplarysearch parameters as follows: Mismatch -2; gap open 5; gap extension 2.For polypeptide sequence comparisons, a BLASTP algorithm is typicallyused in combination with a scoring matrix, such as PAM100, PAM 250,BLOSUM 62 and the like.

[0056] As used herein, the term “isolated,” when used as a modifier ofinvention compositions (e.g., antibodies, subsequences, modified forms,multimers, nucleic acids encoding same, cells, vectors, etc.), meansthat the compositions are made by the hand of man and are separated fromtheir naturally occurring in vivo environment. Generally, compositionsso separated are substantially free of one or more materials with whichthey normally associate with in nature, for example, one or moreprotein, nucleic acid, lipid, carbohydrate, cell membrane. An “isolated”antibody can also be “substantially pure” when free of most or all ofthe materials with which they may normally associate with in nature.Thus, an isolated molecule that also is substantially pure does notinclude polypeptides or polynucleotides present among millions of othersequences, such as antibodies of an antibody library or nucleic acids ina genomic or cDNA library, for example. Purity can be at least about 60%or more by mass. The purity can also be about 70% or 80% or more, andcan be greater, for example, 90% or more. Purity can be determined byany appropriate method, including, for example, UV spectroscopy,chromatography (e.g., HPLC, gas phase), gel electrophoresis (e.g.,silver or coomassie staining) and sequence analysis (nucleic acid andpeptide).

[0057] The invention also provides nucleic acids encoding inventionhumanized antibodies, including high affinity humanized antibodies,subsequences, modified forms and multimers thereof. In variousembodiments, a nucleic acid encodes a polypeptide set forth in SEQ IDNO:1 and 3 (HumA); SEQ ID NO:5 and 7 (HumB); SEQ ID NO:9 and 11 (HumC);SEQ ID NO:13 and 15 (HumD); SEQ ID NO:17 and 19 (HumE); SEQ ID NO:21 and23 (HumF); SEQ ID NO:25 and 27 (HumG); SEQ ID NO:29 and 31 (HumH); andSEQ ID NO:33 and 35 (HumI).

[0058] As used herein, a “nucleic acid” refers to at least two or moreribo- or deoxy-ribonucleic acid base pairs that are linked through aphosphoester bond or equivalent. Nucleic acids include polynucleotidesand polynculeosides. Nucleic acids include single, double or triplex,circular or linear, molecules. A nucleic acid molecule may belongexclusively or in a mixture to any group of nucleotide-containingmolecules, as exemplified by, but not limited to, the following groupsof nucleic acid molecules: RNA, DNA, cDNA, genomic nucleic acids,non-genomic nucleic acids, naturally occurring and non naturallyoccurring nucleic acids and synthetic nucleic acids. This includes, byway of example, nucleic acids associated with any organelle, such as themitochondria, ribosomal RNA, and nucleic acid molecules comprisedchimerically of one or more components that are not naturally occurringalong with naturally occurring components.

[0059] Additionally, a “nucleic acid molecule” may contain in part oneor more non-nucleotide-based components as exemplified by, but notlimited to, amino acids and sugars. Thus, by way of example, but notlimitation, a ribozyme that is in part nucleotide-based and in partprotein-based is considered a “nucleic acid molecule.” Nucleic acids canbe of any length. Nucleic acid lengths typically range from about 20 to10 Kb, 10 to 5 Kb, 1 to 5 Kb or less, 1000 to about 500 base pairs orless in length. Nucleic acids can also be shorter, for example, 100 toabout 500 base pairs, or from about 12 to 25, 25 to 50, 50 to 100, 100to 250, or about 250 to 500 base pairs in length.

[0060] As a result of the degeneracy of the genetic code, nucleic acidsinclude sequences and subsequences degenerate with respect to nucleicacids that encode SEQ ID NO: 1 and 3 (HumA); SEQ ID NO:5 and 7 (HumB);SEQ ID NO:9 and 11 (HumC); SEQ ID NO:13 and 15 (HumD); SEQ ID NO:17 and19 (HumE); SEQ ID NO:21 and 23 (HumF); SEQ ID NO:25 and 27 (HumG); SEQID NO:29 and 31 (HumH); and SEQ ID NO:33 and 35 (HumI), and subsequencesthereof. Nucleic acids also include sequences complementary to asequence that encodes SEQ ID NO: 1 and 3 (HumA); SEQ ID NO:5 and 7(HumB); SEQ ID NO:9 and 11 (HumC); SEQ ID NO:13 and 15 (HumD); SEQ IDNO:17 and 19 (HumE); SEQ ID NO:21 and 23 (HumF); SEQ ID NO:25 and 27(HumG); SEQ ID NO:29 and 31 (HumH); and SEQ ID NO:33 and 35 (HumI), andsubsequences thereof. Nucleic acid subsequences have from about 15 to25, 25 to 50 or 50 to 100 nucleotides. Such nucleic acids are useful forhybridization to detect the presence or an amount of humanized antibodyin a sample (in vitro, cell, culture medium, tissue or organ, serum, ina subject, etc.).

[0061] The invention further includes nucleic acids that hybridize athigh stringency to nucleic acids that encode SEQ ID NO: 1 and 3 (HumA);SEQ ID NO:5 and 7 (HumB); SEQ ID NO:9 and 11 (HumC); SEQ ID NO:13 and 15(HumD); SEQ ID NO:17 and 19 (HumE); SEQ ID NO:21 and 23 (HumF); SEQ IDNO:25 and 27 (HumG); SEQ ID NO:29 and 31 (HumH); and SEQ ID NO:33 and 35(HumI), subsequences thereof and nucleic acid sequences complementarythereto. Hybridizing nucleic acids are also useful for detecting thepresence or an amount of humanized antibody in a sample.

[0062] The term “hybridize” refers to the binding between complementarynucleic acids. Sequences will generally have more than about 50%homology to a nucleic acid that encodes SEQ ID NO: 1 or 3 (HumA); SEQ IDNO:5 or 7 (HumB); SEQ ID NO:9 or 11 (HumC); SEQ ID NO:13 or 15 (HumD);SEQ ID NO:17 or 19 (HumE); SEQ ID NO:21 or 23 (HumF); SEQ ID NO:25 or 27(HumG); SEQ ID NO:29 or 31 (HumH); and SEQ ID NO:33 or 35 (HumI). Theregion between related sequences can extend over at least about 30 basepairs, or about 50 base pairs, or about 100 to 200 or more residues.

[0063] As is understood by those skilled in the art, the T_(M) (meltingtemperature) refers to the temperature at which binding betweencomplementary sequences is no longer stable. For two sequences to bind,the temperature of a hybridization reaction must be less than thecalculated T_(M) for the sequences. The T_(M) is influenced by theamount of sequence complementarity, length, composition (% GC), type ofnucleic acid (RNA vs. DNA), and the amount of salt, detergent and othercomponents in the reaction (e.g., formamide). All of these factors areconsidered in establishing appropriate hybridization conditions (see,e.g., the hybridization techniques and formula for calculating T_(M)described in Sambrook et al., 1989, supra).

[0064] Typically, wash conditions are adjusted so as to attain thedesired degree of hybridization stringency. Thus, hybridizationstringency can be determined empirically, for example, by washing underparticular conditions, e.g., at low stringency conditions or highstringency conditions. Optimal conditions for selective hybridizationwill vary depending on the particular hybridization reaction involved.An example of high stringency hybridization conditions are as follows:2×SSC/0.1% SDS at about 37EC or 42EC (hybridization conditions);0.5×SSC/0.1% SDS at about room temperature (low stringency wash);0.5×SSC/0.1% SDS at about 42EC (moderate stringency wash); and 0.1×SSC/0.1% SDS at about 65EC (high stringency wash).

[0065] Nucleic acids of the invention can be produced using variousstandard cloning and chemical synthesis techniques. Such techniquesinclude, but are not limited to: 1) nucleic acid amplification, e.g.,polymerase chain reaction (PCR), with genomic DNA or cDNA targets usingprimers (e.g., a degenerate primer mixture) capable of annealing toantibody sequence; 2) chemical synthesis of nucleic acid sequences whichcan then be cloned into a plasmid, propagated amplified and purifiedand; 3) computer searches of databases for related sequences. Purity ofnucleic acids can be determined through sequencing, gel electrophoresisand the like.

[0066] The invention further provides expression cassettes comprising anucleic acid encoding a humanized antibody operably linked to anexpression control element. As used herein, the term “operably linked”refers to a physical or a functional relationship between the elementsreferred to that permit them to operate in their intended fashion. Thus,an expression control element “operably linked” to a nucleic acid meansthat the control element modulates transcription and as appropriate,translation of the transcript.

[0067] There need not be physical linkage to nucleic acid in order tocontrol expression. Thus, physical linkage is not required for theelements to be operably linked. For example, a minimal element can belinked to a nucleic acid encoding a humanized antibody. A second elementthat controls expression of an operably linked nucleic acid encoding aprotein that functions “in trans” to bind to the minimal element caninfluence expression of the humanized antibody. Because the secondelement regulates expression of humanized antibody, the second elementis operably linked to the nucleic acid encoding the humanized antibody.

[0068] The term “expression control element” refers to nucleic acid thatinfluences expression of an operably linked nucleic acid. Promoters andenhancers are particular non-limiting examples of expression controlelements. A “promotor sequence” is a DNA regulatory region capable ofinitiating transcription of a downstream (3′ direction) coding sequence.The promoter sequence includes a minimum number of bases necessary toinitiate transcription. Enhancers also regulate gene expression but canfunction a distance from the transcription start site of the gene towhich it is operably linked. Enhancers also function at either 5′ or 3′ends of the gene, as well as within the gene (e.g., in introns or codingsequences).

[0069] An expression control element can confer expression in a mannerthat is “constitutive,” such that transcription of the operably linkednucleic acid occurs without the presence of a signal or stimuli.Expression control elements can confer expression in a manner that is“regulatable,” that is, a signal or stimuli increases or decreasesexpression of the operably linked nucleic acid. A regulatable elementthat increases expression of the operably linked nucleic acid inresponse to a signal or stimuli is also referred to as an “inducibleelement” A regulatable element that decreases expression of the operablylinked nucleic acid in response to a signal or stimuli is referred to asa “repressible element” (i.e., the signal decreases expression such thatwhen the signal, is removed or absent, expression is increased).

[0070] Expression control elements include elements active in aparticular tissue or cell type, referred to herein as a “tissue-specificexpression control elements.” Tissue-specific expression controlelements are typically active in specific cell or tissue because theyare recognized by transcriptional activator proteins, or otherregulators of transcription, that are unique to a specific cell ortissue type.

[0071] Expression control elements additionally include elements thatconfer expression at a particular stage of the cell cycle ordifferentiation. Accordingly, the invention further includes expressioncontrol elements that confer constitutive, regulatable, tissue-specific,cell cycle specific, and differentiation stage specific expression.

[0072] Expression control elements include full-length nucleic acidsequences, such as native promoter and enhancer elements, as well assubsequences or nucleotide variants thereof (e.g., substituted/mutatedor other forms that differ from native sequences) which retain all orpart of full-length or non-variant control element function (conferregulation, e.g., retain some amount of inducibility in response to asignal or stimuli).

[0073] For bacterial systems, constitutive promoters such as T7 and thelike, as well as inducible promoters such as pL of bacteriophage λ,plac, ptrp, ptac (ptrp-lac hybrid promoter) may be used. In insect cellsystems, constitutive or inducible promoters (e.g., ecdysone) may beused. In yeast, constitutive or inducible promoters may be used (see,e.g., Ausubel et al., In: Current Protocols in Molecular Biology, Vol.2, Ch. 13, ed., Greene Publish. Assoc. & Wiley Interscience, 1988; Grantet al., (1987) In: Methods in Enzymology, 153, 516-544, eds. Wu &Grossman, 31987, Acad. Press, N.Y.; Glover, DNA Cloning, Vol. 11, Ch. 3,IRL Press, Wash., D.C., 1986; Bitter (1987) In: Methods in Enzymology,152, 673-684, eds. Berger & Kimmel, Acad. Press, N.Y.; and, Strathem etal., The Molecular Biology of the Yeast Saccharomyces (1982) eds. ColdSpring Harbor Press, Vols. I and II). A constitutive yeast promoter suchas ADH or LEU2 or an inducible promoter such as GAL may be used (R.Rothstein In: DNA Cloning, A Practical Approach, Vol. 11, Ch. 3, ed.D.M. Glover, IRL Press, Wash., D.C., 1986).

[0074] For mammalian cells, constitutive promoters of viral or otherorigins may be used. For example, SV40, or viral long terminal repeats(LTRs) and the like, or inducible promoters derived from the genome ofmammalian cells (e.g., metallothionein IIA promoter; heat shockpromoter, steroid/thyroid hormone/retinoic acid response elements) orfrom mammalian viruses (e.g., the adenovirus late promoter; theinducible mouse mammary tumor virus LTR) can be used for expression.

[0075] The invention also provides transformed cells and progeny thereofinto which a nucleic acid molecule encoding humanized antibody has beenintroduced by means of recombinant DNA techniques in vitro, ex vivo orin vivo. The transformed cells can be propagated and the introducednucleic acid transcribed, or encoded protein expressed. It is understoodthat a progeny cell may not be identical to the parental cell, sincethere may be mutations that occur during replication. Transformed cellsinclude but are not limited to prokaryotic and eukaryotic cells such asbacteria, fingi, plant, insect, and animal (e.g., mammalian, includinghuman) cells. The cells may be present in culture, in a cell, tissue ororgan ex vivo or present in a subject.

[0076] The term “transformed” means a genetic change in a cell followingincorporation of nucleic acid (e.g., a transgene) exogenous to the cell.Thus, a “transformed cell” is a cell into which, or a progeny of which anucleic acid molecule has been introduced by means of recombinant DNAtechniques. Cell transformation to produce host cells may be carried outas described herein or using techniques known in the art. Accordingly,methods of producing cells containing the nucleic acids and cellsexpressing the humanized antibodies of the invention are also provided.

[0077] Typically cell transformation employs a vector. The term“vector,” refers to, e.g., a plasmid, virus, such as a viral vector, orother vehicle known in the art that can be manipulated by insertion orincorporation of a nucleic acid, for genetic manipulation (i.e.,“cloning vectors”), or can be used to transcribe or translate theinserted polynucleotide (i.e., “expression vectors”). Such vectors areuseful for introducing nucleic acids, including a nucleic acid thatencodes a humanized antibody operably linked with an expression controlelement, and expressing the encoded protein in vitro (e.g., in solutionor in solid phase), in cells or in vivo.

[0078] A vector generally contains at least an origin of replication forpropagation in a cell. Control elements, including expression controlelements as set forth herein, present within a vector, are included tofacilitate transcription and translation. The term “expression controlelement” is intended to include, at a minimum, one or more componentswhose presence can influence expression, and can include componentsother than or in addition to promoters or enhancers, for example, leadersequences and fusion partner sequences, internal ribosome binding sites(IRES) elements for the creation of multigene, or polycistronic,messages, splicing signal for introns, maintenance of the correctreading frame of the gene to permit in-frame translation of mRNA,polyadenylation signal to provide proper polyadenylation of thetranscript of a gene of interest, stop codons, etc.

[0079] Vectors can include a selection marker. As is known in the art,“selection marker” means a gene that allows for the selection of cellscontaining the gene. “Positive selection” refers to a process wherebyonly cells that contain the selection marker will survive upon exposureto the positive selection. Drug resistance is one example of a positiveselection marker; cells containing the marker will survive in culturemedium containing the selection drug, and cells which do not contain themarker will die. Such markers include drug resistance genes such as neo,which confers resistance to G418, hygr, which confers resistance tohygromycin, or puro which confers resistance to puromycin, among others.Other positive selection marker genes include genes that allowidentification or screening of cells containing the marker. These genesinclude genes for fluorescent proteins (GFP), the lacZ gene, thealkaline phosphatase gene, and surface markers such as CD8, amongothers.

[0080] Vectors can contain negative selection markers. “Negativeselection” refers to a process whereby cells containing a negativeselection marker are killed upon exposure to an appropriate negativeselection agent. For example, cells which contain the herpes simplexvirus-thymidine kinase (HSV-tk) gene (Wigler et al., Cell 11:223 (1977))are sensitive to the drug gancyclovir (GANC). Similarly, the gpt generenders cells sensitive to 6-thioxanthine.

[0081] Additional selection systems may be used, including, but notlimited to the hypoxanthine-guanine phosphoribosyltransferase gene(Szybalska et al., Proc. Natl. Acad. Sci. USA 48:2026 (1962)), and theadenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980))genes. Additional selectable genes have been described, namely trpB,which allows cells to utilize indole in place of tryptophan; hisD, whichallows cells to utilize histinol in place of histidine (Hartman et al.,Proc. Natl. Acad. Sci. USA 85:8047 (1988)); and ODC (ornithinedecarboxylase), which confers resistance to the ornithine decarboxylaseinhibitor, 2-(difluoromethyl)-DL-omithine, DFMO (McConlogue (1987) In:Current Communications in Molecular Biology, Cold Spring HarborLaboratory, ed.).

[0082] Vectors included are those based on viral vectors, such asretroviral, adeno-associated virus, adenovirus, reovirus, lentivirus,rotavirus genomes, simian virus 40 (SV40) or bovine papilloma virus,etc., modified for introducing and expressing a nucleic acid in a cell(Cone et al., Proc. Natl. Acad. Sci. USA 81:6349 (1984)). (EukaryoticViral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982; Sarveret al., Mol. Cell. Biol. 1:486 (1981)). Additional viral vectors usefulfor expression include parvovirus, rotavirus, Norwalk virus,coronaviruses, paramyxo and rhabdoviruses, togavirus (e.g., sindbisvirus and semliki forest virus) and vesicular stomatitis virus.

[0083] Mammalian expression systems further include vectors specificallydesigned for in vivo and ex vivo expression. Such systems includeadeno-associated virus (AAV) vectors (U.S. Pat. No. 5,604,090). AAVvectors have previously been shown to provide expression of Factor IX inhumans and in mice at levels sufficient for therapeutic benefit (Kay etal., Nat. Genet. 24:257 (2000); Nakai et al., Blood 91:4600 (1998)).Adenoviral vectors (U.S. Pat. Nos. 5,700,470, 5,731,172 and 5,928,944),herpes simplex virus vectors (U.S. Pat. No. 5,501,979) and retroviral(e.g., lentivirus vectors are useful for infecting dividing as well asnon-dividing cells and foamy virues) vectors (U.S. Pat. Nos. 5,624,820,5,693,508, 5,665,577, 6,013,516 and 5,674,703 and WIPO publicationsW092/05266 and W092/14829) and papilloma virus vectors (e.g., human andbovine papilloma virus) have all been employed in gene therapy (U.S.Pat. No. 5,719,054). Vectors also include cytomegalovirus (CMV) basedvectors (U.S. Pat. No. 5,561,063). Vectors that efficiently delivergenes to cells of the intestinal tract have been developed and also maybe used (see, e.g., U.S. Pat. Nos. 5,821,235, 5,786,340 and 6,110,456).

[0084] In yeast, vectors that facilitate integration of foreign nucleicacid sequences into a chromosome, via homologous recombination, forexample, are known in the art and can be used. Yeast artificialchromosomes (YAC) are typically used when the inserted nucleic acids aretoo large for more conventional vectors (e.g., greater than about 12kb).

[0085] Introduction of nucleic acid encoding humanized antibody andhumanized antibody into target cells can also be carried out byconventional methods known in the art such as osmotic shock (e.g.,calcium phosphate), electroporation, microinjection, cell fusion, etc.Introduction of nucleic acid and polypeptide in vitro, ex vivo and invivo can also be accomplished using other techniques. For example, apolymeric substance, such as polyesters, polyamine acids, hydrogel,polyvinyl pyrrolidone, ethylene-vinylacetate, methylcellulose,carboxymethylcellulose, protamine sulfate, or lactide/glycolidecopolymers, polylactide/glycolide copolymers, or ethylenevinylacetatecopolymers. A nucleic acid can be entrapped in microcapsules prepared bycoacervation techniques or by interfacial polymerization, for example,by the use of hydroxymethylcellulose or gelatin-microcapsules, or poly(methylmethacrolate) microcapsules, respectively, or in a colloid drugdelivery system. Colloidal dispersion systems include macromoleculecomplexes, nano-capsules, microspheres, beads, and lipid-based systems,including oil-in-water emulsions, micelles, mixed micelles, andliposomes.

[0086] The use of liposomes for introducing various compositions intocells, including nucleic acids, is known to those skilled in the art(see, e.g., U.S. Pat. Nos. 4,844,904, 5,000,959, 4,863,740, and4,975,282). A carrier comprising a natural polymer, or a derivative or ahydrolysate of a natural polymer, described in WO 94/20078 and U.S. Pat.No. 6,096,291, is suitable for mucosal delivery of molecules, such aspolypeptides and polynucleotides. Piperazine based amphilic cationiclipids useful for gene therapy also are known (see, e.g., U.S. Pat. No.5,861,397). Cationic lipid systems also are known (see, e.g., U.S. Pat.No. 5,459,127). Accordingly, viral and non-viral vector means ofdelivery into cells or tissue, in vitro, in vivo and ex vivo areincluded.

[0087] The invention further provides kits comprising one or morecompositions of the invention, including pharmaceutical formulations,packaged into suitable packaging material. In one embodiment, a kitincludes a humanized antibody or subsequence. In another embodiment, akit includes a nucleic acid encoding humanized antibody or subsequence.In additional embodiments, a kit includes nucleic acids that furtherinclude an expression control element; an expression vector; a viralexpression vector; an adeno-associated virus expression vector; anadenoviral expression vector; and a retroviral expression vector. In yetan additional embodiment, a kit includes a cell that expresses ahumanized antibody or subsequence.

[0088] In additional embodiments, a kit includes a label or packaginginsert including instructions for expressing a humanized antibody or anucleic acid encoding a humanized antibody in cells in vitro, in vivo,or ex vivo. In yet additional embodiments, a kit includes a label orpackaging insert including instructions for treating a subject (e.g., asubject having or at risk of having asthma) with a humanized antibody ora nucleic acid encoding a humanized antibody in vivo, or ex vivo.

[0089] As used herein, the term “packaging material” refers to aphysical structure housing the components of the kit. The packagingmaterial can maintain the components sterilely, and can be made ofmaterial commonly used for such purposes (e.g., paper, corrugated fiber,glass, plastic, foil, ampules, etc.). The label or packaging insert caninclude appropriate written instructions, for example, practicing amethod of the invention, e.g., treating the common cold. Kits of theinvention therefore can additionally include instructions for using thekit components in a method of the invention.

[0090] Instructions can include instructions for practicing any of themethods of the invention described herein. Thus, inventionpharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration to a subject.Instructions may additionally include indications of a satisfactoryclinical endpoint or any adverse symptoms that may occur, or additionalinformation required by the Food and Drug Administration for use on ahuman subject.

[0091] The instructions may be on “printed matter,” e.g., on paper orcardboard within the kit, on a label affixed to the kit or packagingmaterial, or attached to a vial or tube containing a component of thekit. Instructions may comprise voice or video tape and additionally beincluded on a computer readable medium, such as a disk (floppy disketteor hard disk), optical CD such as CD- or DVD-ROM/RAM, magnetic tape,electrical storage media such as RAM and ROM and hybrids of these suchas magnetic/optical storage media.

[0092] Invention kits can additionally include a buffering agent, apreservative, or a protein/nucleic acid stabilizing agent. The kit canalso include control components for assaying for activity, e.g., acontrol sample or a standard. Each component of the kit can be enclosedwithin an individual container or in a mixture and all of the variouscontainers can be within single or multiple packages. For example, aninvention composition can be packaged into a hand pump container orpressurized (e.g., aerosol) container for spraying the composition intothe throat or nasal or sinus passages of a subject.

[0093] The humanized antibodies of the invention, includingsubsequences, modified forms, multimers and nucleic acids encoding them,can be incorporated into pharmaceutical compositions. Suchpharmaceutical compositions are useful for administration to a subjectin vivo or ex vivo, and for providing therapy for a physiologicaldisorder or condition treatable with a humanized antibody.

[0094] Pharmaceutical compositions include “pharmaceutically acceptable”and “physiologically acceptable” carriers, diluents or excipients. Asused herein the terms “pharmaceutically acceptable” and “physiologicallyacceptable” include solvents (aqueous or non-aqueous), solutions,emulsions, dispersion media, coatings, isotonic and absorption promotingor delaying agents, compatible with pharmaceutical administration. Suchformulations can be contained in a liquid; emulsion, suspension, syrupor elixir, or solid form; tablet (coated or uncoated), capsule (hard orsoft), powder, granule, crystal, or microbead. Supplementary activecompounds (e.g., preservatives, antibacterial, antiviral and antifungalagents) can also be incorporated into the compositions.

[0095] Pharmaceutical compositions can be formulated to be compatiblewith a particular local or systemic route of administration. Thus,pharmaceutical compositions include carriers, diluents, or excipientssuitable for administration by particular routes.

[0096] Specific non-limiting examples of routes of administration forcompositions of the invention are inhalation or intranasal delivery.Additional routes include parenteral, e.g., intravenous, intradermal,subcutaneous, oral, transdermal (topical), transmucosal, and rectaladministration.

[0097] Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include: a sterile diluent such as waterfor injection, saline solution, fixed oils, polyethylene glycols,glycerine, propylene glycol or other synthetic solvents; antibacterialagents such as benzyl alcohol or methyl parabens; antioxidants such asascorbic acid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates orphosphates and agents for the adjustment of tonicity such as sodiumchloride or dextrose. pH can be adjusted with acids or bases, such ashydrochloric acid or sodium hydroxide.

[0098] Pharmaceutical compositions for injection include sterile aqueoussolutions (where water soluble) or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, Cremophor EL™ (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof.Fluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Antibacterial andantifungal agents include, for example, parabens, chlorobutanol, phenol,ascorbic acid and thimerosal. Isotonic agents, for example, sugars,polyalcohols such as manitol, sorbitol, sodium chloride can be includedin the composition. Including an agent which delays absorption, forexample, aluminum monostearate and gelatin can prolong absorption ofinjectable compositions.

[0099] Sterile injectable solutions can be prepared by incorporating theactive compound in the required amount in an appropriate solvent withone or a combination of above ingredients followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle containing a basic dispersionmedium and other ingredients as above. In the case of sterile powdersfor the preparation of sterile injectable solutions, methods ofpreparation include, for example, vacuum drying and freeze-drying whichyields a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

[0100] For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays, inhalation devices (e.g.,aspirators) or suppositories. For transdernal administration, the activecompounds are formulated into ointments, salves, gels, or creams asgenerally known in the art.

[0101] Invention humanized antibodies, including subsequences andmodified forms and nucleic acids encoding them, can be prepared withcarriers that protect against rapid elimination from the body, such as acontrolled release formulation or a time delay material such as glycerylmonostearate or glyceryl stearate. The compositions can also bedelivered using implants and microencapsulated delivery systems toachieve local or systemic sustained delivery or controlled release.

[0102] Biodegradable, biocompatable polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to cells or tissues using antibodies or viral coat proteins)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

[0103] Additional pharmaceutical formulations appropriate for thecompositions for administration in the methods of the invention areknown in the art (see, e.g., Remington's Pharmaceutical Sciences (1990)18^(th) ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996)12^(th) ed., Merck Publishing Group, Whitehouse, N.J.; andPharmaceutical Principles of Solid Dosage Forms, Technonic PublishingCo., Inc., Lancaster, Pa., (1993)).

[0104] The pharmaceutical formulations can be packaged in dosage unitform for ease of administration and uniformity of dosage. “Dosage unitform” as used herein refers to physically discrete units suited asunitary dosages for the subject to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the pharmaceuticalcarrier or excipient.

[0105] Humanized antibodies of the invention include antibodies thatprotect against virus infection of cells. For example, HumA, HumB, HumC,HumD, HumF, HumH and HumI protect against HRV infection of cells (FIG.4). Thus, in another embodiment, the invention provides antibodies thatprotect against human rhinovirus (HRV) infection of cells. In oneembodiment, an antibody has a protective efficacy at least 2 to 5 timesgreater than the non-humanized antibody. In another embodiment, anantibody has a protective efficacy at least 5 to 10 times greater thanthe non-humanized antibody. In yet another embodiment, an antibody has aprotective efficacy at least 10 to 20 times greater than thenon-humanized antibody. In still another embodiment, an antibody has aprotective efficacy at least 20 to 30 times greater than thenon-humanized antibody.

[0106] As used herein, “human rhinovirus” or “HRV” means major and minorgroup human serotypes of rhinoviruses that have been identified (see,e.g., Hamparian et al., (1987) Virology 159:191) and those that areidentified later as falling within this class of virus. Major group HRVbinds to ICAM-1 and minor group HRV binds low density lipoprotein (LDL)receptor.

[0107] As used herein, the term “protective efficacy” is the amount ofan antibody which can protect 50% of susceptible cells from infection(i.e. EC₅₀). For example, for HRV, protective efficacy in EC₅₀ is theamount of antibody that protects 50% of hela cells from HRV infection.Thus, a humanized antibody having a protective efficacy 5 times greaterthan another antibody (e.g., non-humanized) can be used in an amount 5fold less than non-humanized antibody while still providing the samedegree of protection from infection.

[0108] Humanized antibodies of the invention include antibodies thatbind to ICAM-1. Although not wishing to be bound by theory, it isbelieved that antibody binding to ICAM-1 inhibits viral binding or theability to infect or penetrate the cell thereby inhibiting viralinfection or proliferation. Such antibodies are therefore useful forinhibiting pathogens such as viruses (e.g., HRV and coxackie A virus,respiratory syncytial virus (RSV)), bacteria, fungi and protozoa (e.g.,malaria) that bind to ICAM-1. Thus, the antibodies are useful forinhibiting HRV infection as well as for inhibiting any microorganism orother pathogens in which ICAM-1 receptor participates. Accordingly, theinvention provides antibodies that inhibit pathogen infection of cellswhere infection is mediated, at least in part, by binding to ICAM-1, andmethods for inhibiting pathogen infection of cells where infection ismediated, at least in part, by binding to ICAM-1.

[0109] In one embodiment, a method includes contacting a virus or cellwith an amount of humanized antibody that binds to ICAM-1 sufficient toinhibit viral infection of the cell. In one aspect, the cell is anepithelial cell. In another embodiment, a method includes administeringto a subject an amount of humanized antibody that binds to ICAM-1sufficient to inhibit viral infection of the subject. In variousaspects, the virus is HRV, coxackie A virus and respiratory syncytialvirus. In yet another embodiment, a method includes administering to asubject an amount of humanized antibody that binds to ICAM-1 sufficientto inhibit infection of the subject by a pathogen.

[0110] The invention also provides methods for inhibiting infection,inhibiting progression or treating a pathogenic infection of a subject.In one embodiment, a method includes administering to a subject havingor at risk of having an HRV infection an amount of humanized antibodysufficient to inhibit, inhibit progression or to treat HRV infection ofthe subject. In another embodiment, a method includes administering to asubject having or at risk of having an coxackie A virus or respiratorysyncytial virus infection an amount of humanized antibody sufficient toinhibit infection, inhibit progression or to treat coxackie A virus orrespiratory syncytial virus infection of the subject. In still anotherembodiment, a method includes administering to a subject having or atrisk of having malaria an amount of humanized antibody sufficient toinhibit, inhibit progression or to treat malaria of the subject. Invarious aspects, a humanized antibody is selected from SEQ ID NO:1 and 3(HumA); SEQ ID NO:5 and 7 (HumB); SEQ ID NO:9 and 11 (HumC); SEQ IDNO:13 and 15 (HumD); SEQ ID NO:17 and 19 (HumE); SEQ ID NO:21 and 23(HumF); SEQ ID NO:25 and 27 (HumG); SEQ ID NO:29 and 31 (HumH); and SEQID NO:33 and 35 (HumI), and antigen binding subsequences thereof.

[0111] The invention further provides methods of decreasing orinhibiting one or more symptoms of a pathogen infection (e.g., caused byHRV, coxackie A virus, respiratory syncytial virus or malaria). In oneembodiment, a method includes administering to a subject having one ormore symptoms associated with HRV, coxackie A virus, respiratorysyncytial virus or malaria an amount of a humanized antibody sufficientto decrease or inhibit one or more symptoms associated with HRV,coxackie A virus, respiratory syncytial virus or malaria in the subject.Symptoms decreased or inhibited include, for example, for HRV, one ormore of fever, congestion, cough, nasal drip, sore throat, and the likeassociated with the common cold. In another embodiment, a methodincludes administering to a subject having otitis media an amount of ahumanized antibody sufficient to decrease or inhibit one or moresymptoms of otitis media in the subject. In yet another embodiment, amethod includes administering to a subject having bronchitis an amountof a humanized antibody sufficient to decrease or inhibit one or moresymptoms of bronchitis in the subject. In still another embodiment, amethod includes administering to a subject having sinusitis an amount ofa humanized antibody sufficient to decrease or inhibit one or moresymptoms of sinusitis in the subject. In a further embodiment, a methodincludes administering to a subject having or at risk of having asthmaan amount of a humanized antibody sufficient to decrease or inhibitasthma exacerbation. In one aspect, the humanized antibody isadministered locally. In another aspect, the humanized antibody isadministered via inhalation or intranasaly.

[0112] In addition to inhibiting pathogens that function directly orindirectly through ICAM-1, invention humanized antibodies can be used totreat undesirable physiological conditions, such as disease or disordersin which ICAM-1 plays a role. For example, LFA-1 interaction with ICAM-1participates in inflammation. Thus, an invention antibody may be used toinhibit this interaction thereby modulating (e.g., decrease) local orsystemic inflammation. Furthermore, ICAM-1 plays a role in other immuneresponse pathways, cancer and metastasis. Thus, an invention antibodymay be used to reduce or prevent organ transplant rejection orautoimmune diseases or cancer or metastasis. Accordingly, the inventionprovides antibodies that modulate immune responsiveness (e.g.,inflammation) and other cellular processes in which ICAM-1 participatesand methods for modulating immune response pathways.

[0113] The methods of the invention may be practiced prior to infection(i.e. prophylaxis) or after infection, before or after acute or chronicsymptoms of the infection or physiological condition or disorderdevelops (e.g., before organ transplantation). Administering acomposition prior to or immediately following development of symptomsmay lessen the severity of the symptoms in the subject. Administering acomposition prior to development of symptoms in the subject may decreasecontagiousness of the subject thereby decreasing the likelihood of othersubjects becoming infected from the infected subject.

[0114] The term “subject” refers to animals, typically mammaliananimals, such as a non-human primate (gorillas, chimpanzees, orangutans,macaques, gibbons), a domestic animal (dogs and cats), a farm animal(horses, cows, goats, sheep, pigs), experimental animal (mouse, rat,rabbit, guinea pig) and humans. Human subjects include adults, andchildren, for example, newborns and older children, for example, betweenthe ages of 1 and 5, 5 and 10 and 10 and 18. Human subjects may includethose having or at risk of having a viral infection, such as HRV, andwhich develops one or more symptoms of the infection, for example, thosetypically associated with the common cold. Human subjects include thosehaving or at risk of having asthma, including asthmatics suffering fromchronic asthma prior to or following suffering an acute asthma attack.Subjects include disease model animals (e.g., such as mice and non-humanprimates) for testing in vivo efficacy of humanized antibodies of theinvention (e.g., an HRV animal model, an asthma animal model, an organtransplant model, an autoimmune disorder model, cancer model, etc.).

[0115] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described herein.

[0116] All publications, patents and other references cited herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control.

[0117] As used herein, the singular forms “a”, “and,” and “the” includeplural referents unless the context clearly indicates otherwise. Thus,for example, reference to “a transformed cell” includes a plurality ofsuch cells and reference to “a humanized antibody” can include referenceto one or more such cells or antibodies, and so forth.

[0118] A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, the following examples are intended to illustrate but notlimit the scope of invention described in the claims.

EXAMPLES Example 1

[0119] This example describes the strategy for humanizing 1A6.

[0120] Mouse monoclonal antibody 1A6 (mAb1A6) was developed by Colonnoet al., and has been shown to bind specifically to ICAM-1 and protectcells against infection by human rhinovirus (HRV) major group (Colonno RJ, et al. (1991) European Patent Application #91201243.2; Publicationnumber: 0 459 577 A2, which also describes the sequence of mousemAb1A6). The parental mouse monoclonal antibody 1A6 was synthesized inthe form of scFv. The purified protein, MsclA6, has an affinity of1.18×10⁻⁶ M in K_(D) against ICAM-1 (Table 4).

[0121] To humanize mAb1A6, selected human VH subgroup III and VL-kappasubgroup I consensus sequences were selected as the acceptor VH and VLframeworks, respectively (Padlan (1994) Molecular Immunol. 31:169-217;Padlan (1991) Molecular Immunol. 28:489-498). These human sequences havepreviously been used to humanize two antibodies (Carter et al. (1992)Proc. Natl. Acad. Sci. USA 89:4285-4289; Presta et al. (1993) J.Immunol. 151:2623-2632).

[0122] Among a total of 82 amino acid residues in the heavy chainframework, the human VH III consensus sequence and mAblA6 antibody share56 identical amino acid residues, which amounts to 68.3% identity. Among81 light chain framework residues, the human κ I consensus sequence andmAb1A6 antibody have 52 amino acid residues in common, which equals to64.2% identity. (FIG. 1).

[0123] Among a total of 55 framework amino acid residues that aredifferent between mAb1A6 and human consensus sequences, 49 of them areeither located on the surface of the antibody molecule, or are residueswith similar characteristics, therefore human consensus residues can beused to replace mouse residues. The remaining six positions, VH 37, 69,71, 73, 94 and VL 49, belong to the “Vernier” zone as described by Tooteand Winter (1992, J. Mol. Biol. 224:487-499). Because “Vernier” zoneresidues form a layer underlying the CDRs and may impact on thestructure of CDRs and the affinity of the antibody, residues at thesepositions were chosen based on molecular model building of the antibody(FIG. 2).

[0124] VL 49

[0125] Inspection reveals that this position is both at the center ofthe antibody combining site and at the light chain/heavy chaininterface. Substituting an ideal residue at this position can improveantigen binding by both providing additional direct binding contact andby improving the character of the interface. Tyrosine, found in humanantibodies at this position, does both. Model building suggests that Y49can form both Van der Waals and H bond contact with ICAM. Y49 also caninteract with heavy chain W102, completing a network of interactingaromatic residues that provide both binding interaction and flexibilityat the light chain/heavy chain interface. Therefore, human consensusresidue tyrosine at this position is superior to the parental mouseresidue lysine.

[0126] VH 37

[0127] This residue is at the interface between the light and heavychains. Comparing to the parental mouse residue methionine, the humanconsensus residue valine intrudes less on the interface, potentiallyproviding additional flexibility. Flexibility at the interface canenhance binding affinity by increasing conformational adaptability ofthe antibody. (FIG. 2C)

[0128] VH 69

[0129] This residue is packed in the interior of the variable domain.The murine residue, methionine, makes a potentially destabilizingcontact with the backbone of a neighboring beta strand. In contrast, thehuman residue isoleucine packs well in the interior of the protein.

[0130] VH 73

[0131] Molecular modeling indicates that the human consensus residue,aspartic acid (D73) can interact with K30 of heavy chain CDR1. Sincemodel building suggests that K30 is not involved directly in antigenbinding, this stabilizing change is predicted to be either neutral orbeneficial.

[0132] VH 71 and VH94

[0133] Structural inspection indicated that both of these positionsrequire a residue with a small side chain for maintenance of properantibody conformation. Therefore, the human consensus residue at thisposition, arginine, is not appropriate. Serine and glycine were selectedfor position 71.

[0134] According to Chothia et al., residues at VH94 is related with thecanonical structure of H1 or CDR1 (defined as VH26-VH32). The CDR1 of1A6 belongs to the canonical structure 1 and family 1 (Chothia and Lesk(1987) J. Mol. Biol. 186:651-663; Chothia et al. (1992) J. Mol. Biol.227:799-817; Chothia et al. (1989) Nature 342:877-883). Corresponding tothis canonical structure, human sequences showed three possible residuesat VH94 position: arginine, threonine or alanine (Chothia et al. (1992)J. Mol. Biol. 227:799-817). Since arginine is not appropriate for thisparticular antibody, alanine, threonine and another small residue,aspartic acid were chosen.

[0135] Finally, molecular model building indicates that a portion of theCDR2 in the VH domain, VH60-64, does not have direct contact with theantigen. Therefore mouse residues at these positions (DPKVQ) can bereplaced by human residues ADSVK.

Example 2

[0136] This example describes the preparation of several humanized scFvexpression constructs.

[0137] The humanized scFv A (HumA) cDNA (FIG. 4) containing 750 bb wassynthesized using a series of overlapping oligonucleotides. Theseoverlapping oligonucleotides (Table 1) were designed to encode the aminoacids of the variable region of the heavy (VH) and light (VL) chainslinked by a linker((G₄S)₄) with a Bam H1 site. The heavy chain and lightchain were cloned separately in TOPO 2.1 vector. After DNA sequencingconformation, the heavy and light chain were subcloned into expressionvector (pBAD/pIII A) to form full length DNA.

[0138] The oligonucleotides were first annealed in six groups consistingof oligo AVH1/AVH2, oligo AVH3/AVH4, oligo AVH5/AVH6 for heavy chain,and oligo AVL1/AVL2, oligo AVL3/AVL4, oligo AVL5/AVL6 for the lightchain. Each annealed group was extended with the Klenow fragment of DNApolymerase. The annealed and extended products of group 1-3 were pooledwith oligo AVH7 as overlapping templates that were amplified viapolymerase chain reaction (PCR) using the high-fidelity thermostable DNApolymerase (Roche) with oligo AVH8 and AVH9 as primers. The annealed andextended products of group 4-6 were pooled with oligo AVL7 asoverlapping templates that were also amplified via polymerase chainreaction (PCR) using oligo AVL8 and AVL9 as primers. The PCR productswere directly inserted into the TA cloning vector pCR2.1-TOPO(Invitrogen) and transferred into TOP10 competent cells. The plasmidswith inserts were isolated and sequenced.

[0139] The light chain and the heavy chain DNA fragments were isolatedfrom their cloning vector by digestion with Nco I/Bam HI and Bam HI/VHpaI respectively, and cloned into expression vector pBAD/pIII A cuttingwith Nco I/Sal I (blunted) to be in frame with the carboxy-terminal Histag. Both strands of the expression construct pBAD-HumA was sequenced(MWG Biotech, Inc.).

[0140] All other human scFv expression constructs (HumB to H) were madewith the same procedure as HumA described above except using differentoligonucleotides (Table 1).

[0141] For HumB, using BVH6 and BVH7 to replace AVH6 and AVH7; for HumC,using CVH5, CVH6 and CVH7 to replace AVH5, AVH6 and AVH7; for HumD,using DVH6 and DVH7 to replace AVH6 and AVH7; for HumE, using EVH4,EVH5, EVH6 and EVH7 to replace AVH4, AVH5, AVH6 and AVH7; for HumF,using FVH6 and FVH7 to replace AVH6 and AVH7; for HumG, using GVL3,GVL4, GVH5, GVH6 and GVH7 to replace AVL3, AVL4, AVH5, AVH6 and AVH7;for HumH, using HVL3, HVL4, HVH4, HVH5, HVH6 and HVH7 to replace AVL3,AVL4, AVH4, AVH5, AVH6 and AVH7; for HumI, using IVL3, IVL4, IVH4, IVH5,IVH6 and IVH7 to replace AVL3, AVL4, AVH4, AVH5, AVH6 and AVH7. TABLE 1Oligonucleotides for humanized scFvs Oligonucleotides for the light(V_(H)) chain of HumA: AVL-1:CGAACCATGGGCGATATCCAGATGACCCAATCTCCGTCTAGCCTGAGCGCCAGTGTTGGTG AVL-2:GTGAAGATTATTACTGATAGATTGGCTGGCGCGGCAAGTAATGGTAACTCGATCACCAACACTGGCGCTCAG AVL-3:CTATCAGTAATAATCTTCACTGGTATCAACAAAAACCGGGTAAAGCTCCGAAACTTCTTATCT ATCACGCCAVL-4: CCCGAGCCAGAGCCAGAGAAGCGGCTCGGAACGCCGCTAATGCTCTGAGAGGCGTGATAGATAAGAAG AVL-5:CTCTGGCTCTGGCTCGGGCACGGACTTTACCCTTACCATCAGCTCTCTTCAGCCGGAAGAC TTTGCCACCAVL-6: CCTTGACCGAAGGTATACGGCCAGCTATTAGACTGCTGACAATAATAGGTGGCAAAGTCTTCCGGC AVL-7:GTATACCTTCGGTCAAGGTACCAAGGTCGAGATTAAGCGCGGCGGTGGCGGTTCTGGTGGC GGTGGTAGCGAVL-8: CGAACCATGGGCGATATCCAGATGACCCAATC AVL-9:CGGATCCACCGCCACCGCTACCACCGCCACCAG Oligonucleotides for the heavy (V_(H))chain of HumA: AVH-1:GGTGGCGGTGGATCCGGTGGCGGTGGCAGCGAAGTTCAACTTGTTGAGTCTGGTGGCGGTCTGGTTCAGCCGG AVH-2:GTCCTTAATGTTGAAACCGCTTGCTGCGCAAGACAGGCGCAGAGAGCCACCCGGCTGAACC AGACCGCCACAVH-3: GGTTTCAACATTAAGGACACCTACATCCATTGGGTGAGGCAAGCTCCGGGTAAGGGTCTGGAGTGGG AVH-4:GGCCCTTCACGCTGTCAGCGTAAATGGTGTTGTCGTTTGCCGGGTCGATACGTGCCACCCACTCCAGACCCTTACC AVH-5:CGCTGACAGCGTGAAGGGCCGTTTTACTATTTCTAGCGACGACTCTAAGAACACCGCGTACCTTCAGATGAACTCTCTGCG AVH-6:CCAGTAGCCAGAGTCCGTGCAGTAGTAGACGGCGGTGTCCTCGGCACGCAGAGAGTTCAT CTGAAGGAVH-7: GGACTCTGGCTACTGGTTTGCCTACTGGGGCCAGGGCACGCTTGTCACCGTCTCTTCTGGTTAAC AVH-8: GGTGGCGGTGGATCCGGT AVH-9: GGGTTAACCAGAAGAGACGGOligonucleotides for making other human scFv (Hum B-I): BVH-6:CCAGTAGCCAGAGGCCGTGCAGTAGTAGACGGCGGTGTCCTCGGCACGCAGAGAGTTCAT CTGAAGGBVH-7: GGCCTCTGGCTACTGGTTTGCCTACTGGGGCCAGGGCACGCTTGTCACCGTCTCTTCTGGTTAAC CVH-5:CGCTGACAGCGTACAGGGCCGTTTTACTATTTCTGGCGACGACTCTAAGAACACCGCGTACCTTCAGATGAACTCTCTGCG CVH-6:CCAGTAGCCAGAGGTCGTGCAGTAGTAGACGGCGGTGTCCTCGGCACGCAGAGAGTTCAT CTGAAGGCVH-7: GACCTCTGGCTACTGGTTTGCCTACTGGGGCCAGGGCACGCTTGTCACCGTCTCTTCTGGTTAAC DVH-6: CCAGTAGCCAGAGGTCGTGCAGTAGTAGACGGCGGTGTCCTCGGCACGCAGAGAGTTCATCTGAAGG DVH-7:GACCTCTGGCTACTGGTTTGCCTACTGGGGCCAGGGCACGCTTGTCACCGTCTCTTCTGGT TAACEVH-4: GGCCCTGCACCTTCGGATCGTAAATGGTGTTGTCGTTTGCCGGGTCGATACGTGCCACCCACTCCAGACCCTTACC EVH-5:CGATCCGAAGGTGCAGGGCCGTTTTACTATTTCTGCGGACGACTCTAAGAACACCGCGTACCTTCAGATGAACTCTCTGCG EVH-6:CCAGTAGCCAGAGGTCGTGCAGTAGTAGACGGCGGTGTCCTCGGCACGCAGAGAGTTCAT CTGAAGGEVH-7: GACCTCTGGCTACTGGTTTGCCTACTGGGGCCAGGGCACGCTTGTCACCGTCTCTTCTGGTTAAC FVH-6: CCAGTAGCCAGAGGTCGTGCAGTAGTAGACGGCGGTGTCCTCGGCACGCAGAGAGTTCATCTGAAGG FVH-7:GACCTCTGGCTACTGGTTTGCCTACTGGGGCCAGGGCACGCTTGTCACCGTCTCTTCTGGT TAACGVL-3: CTATCAGTAATAATCTTCACTGGTATCAACAAAAACCGGGTAAAGCTCCGAAACTTCTTATCAAACACGCC GVL-4:CCCGAGCCAGAGCCAGAGAAGCGGCTCGGAACGCCGCTAATGCTCTGAGAGGCGTGAAAG ATAAGAAGGVH-5: CGCTGACAGCGTGAAGGGCCGTTTTACTATTTCTGCGGACGACTCTAAGAACACCGCGTACCTTCAGATGAACTCTCTGCG GVH-6:CCAGTAGCCAGAGGTCGTGCAGTAGTAGACGGCGGTGTCCTCGGCACGCAGAGAGTTCAT CTGAAGGGVH-7: GACCTCTGGCTACTGGTTTGCCTACTGGGGCCAGGGCACGCTTGTCACCGTCTCTTCTGGTTAAC HVL-3:CTATCAGTAATAATCTTCACTGGTATCAACAAAAACCGGGTAAAGCTCCGAAACTTCTTATCA AACACGCCHVL-4: CCCGAGCCAGAGCCAGAGAAGCGGCTCGGAACGCCGCTAATGCTCTGAGAGGCGTGAAAGATAAGAAG HVH-4:GGCCCTGCACCTTCGGATCGTAAATGGTGTTGTCGTTTGCCGGGTCGATACGTGCCACCCACTCCAGACCCTTACC HVH-5:CGATCCGAAGGTGCAGGGCCGTTTTACTATTTCTGCGGACGACTCTAAGAACACCGCGTACCTTCAGATGAACTCTCTGCG HVH-6:CCAGTAGCCAGAGGTCGTGCAGTAGTAGACGGCGGTGTCCTCGGCACGCAGAGAGTTCAT CTGAAGGHVH-7: GACCTCTGGCTACTGGTTTGCCTACTGGGGCCAGGGCACGCTTGTCACCGTCTCTTCTGGTTAAC IVL-3:CTATCAGTAATAATCTTCACTGGTATCAACAAAAACCGGGTAAAGCTCCGAAACTTCTTATCA AACACGCCIVL-4: CCCGAGCCAGAGCCAGAGAAGCGGCTCGGAACGCCGCTAATGCTCTGAGAGGCGTGAAAGATAAGAAG IVH-4:GGCCCTGCACCTTCGGATCGTAAATGGTGTTGTCGTTTGCCGGGTCGATACGTGCCACCCACTCCAGACCCTTACC IVH-5:CGATCCGAAGGTGCAGGGCCGTTTTACTATGTCTGCGGACACCTCTAAGAACACCGCGTACCTTCAGATGAACTCTCTGCG IVH-6:CCAGTAGCCAGAGGTCGTGCAGTAGTAGACGGCGGTGTCCTCGGCACGCAGAGAGTTCAT CTGAAGGIVH-7: GACCTCTGGCTACTGGTTTGCCTACTGGGGCCAGGGCACGCTTGTCACCGTCTCTTCTGGTTAAC

[0142] Molecular model building enabled synthesis of 9 versions ofhumanized antibodies in the form of scFv (HumA-HumI, summarized inTables 2 and 3). Four of the humanized antibodies, HumA-HumD, do nothave parental mouse framework residues, and five of them, HumE-HumI,contain various number of parental mouse residues in the framework. Thesequence of HumB is compared against parental mouse 1A6 and humanconsensus framework in FIG. 3. TABLE 2 Humanization Constructs PositionL49 H37 H60-64 H69 H71 H73 H94 Human/Mouse Y/K V/M ADSVK/DPKVQ I/M R/AD/T R/T HumA Y V ADSVK I S D D HumB Y V ADSVK I S D A HumC Y V ADSVK I GD T HumD Y V ADSVK I S D T HumE Y V DPKVQ I A D T HumF Y V ADSVK I A D THumG K V ADSVK I A D T HumH K V DPKVQ I A D T HumI K M DPKVQ M A T T

[0143] TABLE 3 Amino Acid Sequences of Humanized Antibody Hum A: vHDomain Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser (Gly Phe Asn Ile Lys Asp Thr Tyr IleHis) Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala (Arg IleAsp Pro Ala Asn Asp Asn Thr Ile Tyr Ala Asp Ser Val Lys Gly) Arg Phe ThrIle Ser Ser Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu ArgAla Glu Asp Thr Ala Val Tyr Tyr Cys Thr Asp (Ser Gly Tyr Trp Phe AlaTyr) Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser VL Domain Asp Ile GlnMet Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr IleThr Cys (Arg Ala Ser Gln Ser Ile Ser Asn Asn Leu His) Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr (His Ala Ser Gln Ser IleSer) Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr LeuThr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys (Gln Gln SerAsn Ser Trp Pro Tyr Thr) Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg HumB: VH Domain Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly Ser Leu Arg Leu Ser Cys Ala Ala Ser (Gly Phe Asn Ile Lys Asp Thr TyrIle His) Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala (ArgIle Asp Pro Ala Asn Asp Asn Thr Ile Tyr Ala Asp Ser Val Lys Gly) Arg PheThr Ile Ser Ser Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser LeuArg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ala (Ser Gly Tyr Trp Phe AlaTyr) Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser VL Domain Asp Ile GlnMet Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr IleThr Cys (Arg Ala Ser Gln Ser Ile Ser Asn Asn Leu His) Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr (His Ala Ser Gln Ser IleSer) Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr LeuThr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys (Gln Gln SerAsn Ser Trp Pro Tyr Thr) Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg HumC: VH Domain Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly Ser Leu Arg Leu Ser Cys Ala Ala Ser (Gly Phe Asn Ile Lys Asp Thr TyrIle His) Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala (ArgIle Asp Pro Ala Asn Asp Asn Thr Ile Tyr Ala Asp Ser Val Lys Gly) Arg PheThr Ile Ser Gly Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser LeuArg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Thr (Ser Gly Tyr Trp Phe AlaTyr) Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser VL Domain Asp Ile GlnMet Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr IleThr Cys (Arg Ala Ser Gln Ser Ile Ser Asn Asn Leu His) Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr (His Ala Ser Gln Ser IleSer) Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr LeuThr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys (Gln Gln SerAsn Ser Trp Pro Tyr Thr) Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg HumD: VH Domain Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly Ser Leu Arg Leu Ser Cys Ala Ala Ser (Gly Phe Asn Ile Lys Asp Thr TyrIle His) Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala (ArgIle Asp Pro Ala Asn Asp Asn Thr Ile Tyr Ala Asp Ser Val Lys Gly) Arg PheThr Ile Ser Ser Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser LeuArg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Thr (Ser Gly Tyr Trp Phe AlaTyr) Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser VL Domain Asp Ile GlnMet Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr IleThr Cys (Arg Ala Ser Gln Ser Ile Ser Asn Asn Leu His) Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr (His Ala Ser Gln Ser IleSer) Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr LeuThr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys (Gln Gln SerAsn Ser Trp Pro Tyr Thr) Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg HumE: VH Domain Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly Ser Leu Arg Leu Ser Cys Ala Ala Ser (Gly Phe Asn Ile Lys Asp Thr TyrIle His) Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala (ArgIle Asp Pro Ala Asn Asp Asn Thr Ile Tyr Asp Pro Lys Val Gln Gly) Arg PheThr Ile Ser Ala Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser LeuArg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Thr (Ser Gly Tyr Trp Phe AlaTyr) Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser VL Domain Asp Ile GlnMet Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr IleThr Cys (Arg Ala Ser Gln Ser Ile Ser Asn Asn Leu His) Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr (His Ala Ser Gln Ser IleSer) Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr LeuThr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys (Gln Gln SerAsn Ser Trp Pro Tyr Thr) Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg HumF: VH Domain Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly Ser Leu Arg Leu Ser Cys Ala Ala Ser (Gly Phe Asn Ile Lys Asp Thr TyrIle His) Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala (ArgIle Asp Pro Ala Asn Asp Asn Thr Ile Tyr Ala Asp Ser Val Lys Gly) Arg PheThr Ile Ser Ala Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser LeuArg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Thr (Ser Gly Tyr Trp Phe AlaTyr) Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser VL Domain Asp Ile GlnMet Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr IleThr Cys (Arg Ala Ser Gln Ser Ile Ser Asn Asn Leu His) Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr (His Ala Ser Gln Ser IleSer) Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr LeuThr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys (Gln Gln SerAsn Ser Trp Pro Tyr Thr) Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg HumG: VH Domain Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly Ser Leu Arg Leu Ser Cys Ala Ala Ser (Gly Phe Asn Ile Lys Asp Thr TyrIle His) Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala (ArgIle Asp Pro Ala Asn Asp Asn Thr Ile Tyr Ala Asp Ser Val Lys Gly) Arg PheThr Ile Ser Ala Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser LeuArg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Thr (Ser Gly Tyr Trp Phe AlaTyr) Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser VL Domain Asp Ile GlnMet Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr IleThr Cys (Arg Ala Ser Gln Ser Ile Ser Asn Asn Leu His) Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Leu Leu Ile Lys (His Ala Ser Gln Ser IleSer) Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr LeuThr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys (Gln Gln SerAsn Ser Trp Pro Tyr Thr) Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg HumH: VH Domain Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly Ser Leu Arg Leu Ser Cys Ala Ala Ser (Gly Phe Asn Ile Lys Asp Thr TyrIle His) Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala (ArgIle Asp Pro Ala Asn Asp Asn Thr Ile Tyr Asp Pro Lys Val Gln Gly) Arg PheThr Ile Ser Ala Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser LeuArg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Thr (Ser Gly Tyr Trp Phe AlaTyr) Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser VL Domain Asp Ile GlnMet Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr IleThr Cys (Arg Ala Ser Gln Ser Ile Ser Asn Asn Leu His) Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Leu Leu Ile Lys (His Ala Ser Gln Ser IleSer) Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr LeuThr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys (Gln Gln SerAsn Ser Trp Pro Tyr Thr) Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg HumI: VH Domain Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly Ser Leu Arg Leu Ser Cys Ala Ala Ser (Gly Phe Asn Ile Lys Asp Thr TyrIle His) Trp Met Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala (ArgIle Asp Pro Ala Asn Asp Asn Thr Ile Tyr Asp Pro Lys Val Gln Gly) Arg PheThr Met Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser LeuArg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Thr (Ser Gly Tyr Trp Phe AlaTyr) Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser VL Domain Asp Ile GlnMet Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr IleThr Cys (Arg Ala Ser Gln Ser Ile Ser Asn Asn Leu His) Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Leu Leu Ile Lys (His Ala Ser Gln Ser IleSer) Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr LeuThr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys (Gln Gln SerAsn Ser Trp Pro Tyr Thr) Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

[0144] The CDR Residues are included within brackets.

Example 3

[0145] This example describes expression and purification of humanized1a6 single chain antibodys.

[0146] For production of the humanized 1A6 scFv, TOP10 cells transformedwith disired expression construct were grown in shaker flaks in TBmedium (B10 101) until they reached an OD₆₀₀ of 0.8. Protein expressionwas induced with 0.02% arabinose for eighteen hours at room temperature.Cells were pelleted by cenulfugation at 4,000 g for 15 minutes. Cellpellets were resuspended in {fraction (1/50)}^(th) volume of lysisbuffer (20 mM sodium phosphate, 1% Triton X-100, 500 mM NaCl, 40 mMimidazole, 2 mM 2-mercaptoethanol), 0.2 mM PMSF, 1 mg/ml lysozyme andincubated on ice for 30 minutes. The cell suspension was sonicated andanother aliquot of PMSF was added. The cell debris was pelleted bycentrifugation at 12,000× g and the clarified sonicate was filtered andfractionated by metal affinity chromatography. Induced histidine-taggedproteins were bound to a Hi Trap™ metal chelating column(Amersham/Pharmacia) equilibrated with Ni²⁺ according to themanufacturer's instructions. The column was then washed with four columnvolumes of buffer consisting of 100 mM imidazole, 20 mM sodiumphosphate, pH 7.4, 500 mM NaCl. Fractions of proteins eluted from thecolumn in 500 mM Imidazole, 20 mM sodium phosphate, pH 7.4 werecollected, pooled and dialyzed at 4° C. against phosphate bufferedsaline (PBS)/2 mM EDTA, then dialyzed against PBS.

Example 4

[0147] This example describes studies measuring binding affinity ofhumanized single chain antibody proteins for ICAM-1.

[0148] To evaluate the binding affinity of histidine-tagged human singlechain (hsc) proteins soluble ICAM was used in an ELISA assay. A 96-wellEIA plate (Corning, Inc.) was coated with 100 μl/well soluble ICAM-1(Bender MedSystems) at 1 μg/ml in 0.1 M NaHCO₃. After washing with TBST(50 mM Tris, pH8.0, 150 mM NaCl, 0.05% Tween-20), the plate was blockedwith 3% non-fat milk in TBST at 37° C. for 1 hour. After washing withTBST, the plate was incubated with scFv samples (100 μl /well) dilutedin 1% non-fat milk/TBST solution at room temperature for 1 hour. Afterwashing with TBST, the horse radish peroxidase-conjugated anti-His(C-term) antibody (Invitrogen) diluted 1:2000 in 1% non-fat milk/TBSTwas added and the plate was incubated at room temperature for 1 hour.The plate was washed thoroughly with TBST and 100 μl/well 3,3′,5,5′-tetramethybenzidine substrate solution (Kirkegaard and PerryLaboratories) was added. After 5 min incubation, the color developmentwas stopped by adding 100 ml/well 0.12 N HCl and the absorbance of thewells at 450 nm was measured by a plate reader (ICN).

[0149] Binding studies revealed that all of the humanized scFv proteins(hsc) demonstrate greater than ten times higher binding affinity forICAM-1 than the parental mouse scFv (Table 4). TABLE 4 Mouse 1A6 scFvand Humanized 1A6 scFv ScFv K_(D) (M) EC 50 (μM)* Msc1A6 1.18 × 10⁻⁶ >10HscA 1.50 × 10⁻⁷ 2.8 HscB 2.62 × 10⁻⁸ 0.19 HscC 5.80 × 10⁻⁸ 0.22 HscD2.33 × 10⁻⁸ 0.05 HscF 4.60 × 10⁻⁸ 0.29 HscH 2.09 × 10⁻⁸ 4.2 HscI 1.50 ×10⁻⁷ >10

Example 5

[0150] This example describes data demonstrating that humanized 1A6antibodies protect against HRV infection. This example also describesdata that demonstrate that protection was significantly greater thanmouse 1A6 antibody.

[0151] HeLa cells were plated at 1×10⁵ cells per well of a 48-welltissue culture dish and cultured for 24 hours. Culture medium wasaspirated and 100 μl of humanized 1A6 proteins was added to each well atthe dilution indicated. The plates were incubated for one hour in a 37°C. incubator, the protein solution removed, 200 μl HRV15 (at MOI of 1)was added and the plates incubated for one hour at 33° C. The cells werethen washed and 1 ml/well growth medium added. The infected cells wereincubated at 33° C. for 48 hours. The medium was then aspirated and theremaining viable cells stained with crystal violet. Finally, the crystalviolet was extracted with 2 ml methanol per well, and the extractedstain determined by measuring the A₅₇₀. The percentage protection wascalculated for each point in triplicate using the formula:${\% \quad {protection}} = \frac{(100)\left( {{{Absorbance}\quad {of}\quad {sample}} - {{Absorbance}\quad {of}\quad {virus}\quad {only}}} \right)}{\left( {{{Absorbance}\quad {of}\quad {uninfected}\quad {cells}} - {{Absorbance}\quad {of}\quad {virus}\quad {only}}} \right)}$

[0152] The protection efficacy was quantified as EC₅₀, which is the doseof an antibody protein which can protect 50% of hela cells from HRVinfection. EC₅₀ of several humanized 1A6 proteins are summarized inTable 2, and the data from this protection assay is shown in FIG. 4.This assay revealed that the EC50 of Hum19 scFv protein was more thansixty times higher than that of the parental mouse 1A6 scFv protein(FIG. 4). In vitro protection results correlate well with the antibodybinding affinity.

What is claimed is:
 1. A humanized antibody that binds ICAM-1, saidantibody selected from: SEQ ID NO:1 and 3 (HumA); SEQ ID NO:5 and 7(HumB); SEQ ID NO:9 and 11 (HumC); SEQ ID NO:13 and 15 (HumD); SEQ IDNO:17 and 19 (HumE); SEQ ID NO:21 and 23 (HumF); SEQ ID NO:25 and 27(HumG); SEQ ID NO:29 and 31 (HumH); and SEQ ID NO:33 and 35 (HumI).
 2. Asubsequence of the antibody of claim 1, said antibody subsequencecapable of binding an ICAM-1 epitope.
 3. The humanized antibody of claim2, wherein the antibody subsequence comprises a single chain, Fab, Fab′or (Fab)₂ fragment.
 4. The humanized antibody of claim 1, said antibodyhaving one or more amino acid substitutions, provided that said antibodyis capable of binding an ICAM-1 epitope.
 5. A humanized antibody thatbinds ICAM-1 and inhibits pathogen infection of cells expressing ICAM-1.6. The humanized antibody of claim 5, said antibody having a protectiveefficacy at least 2 times greater than the non-humanized antibody. 7.The humanized antibody of claim 5, said antibody having a protectiveefficacy at least 5 times greater than the non-humanized antibody. 8.The humanized antibody of claim 5, said antibody having a protectiveefficacy at least 10 times greater than the non-humanized antibody. 9.The humanized antibody of claim 5, said antibody having a protectiveefficacy at least 20 times greater than the non-humanized antibody. 10.The humanized antibody of claim 5, said antibody having a protectiveefficacy at least 30 times greater than the non-humanized antibody. 11.The humanized antibody of claim 5, wherein the pathogen is humanrhinovirus (HRV).
 12. The humanized antibody of claim 5, wherein thepathogen is coxackie A virus, respiratory syncytial virus, or malaria.13. The humanized antibody of claim 5, wherein the antibody is an intactimmunoglobulin molecule comprising 2 full-length heavy chains and 2full-length light chains.
 14. The humanized antibody of claim 5, whereinthe antibody is an antibody subsequence that binds to ICAM-1.
 15. Thehumanized antibody of claim 14, wherein the antibody subsequencecomprises a single chain, Fab, Fab′ or (Fab)₂ fragment.
 16. Thehumanized antibody of claim 5, wherein the antibody is multispecific ormultifunctional.;
 17. The humanized antibody of claim 5, wherein theantibody is linked to one or more identical or different antibodies toform a multimer.
 18. The humanized antibody of claim 17, wherein themultimer comprises a homo- or hetero-dimer, trimer, or tetramer.
 19. Thehumanized antibody of claim 17, wherein the multimer is formed via amultimerization domain.
 20. The humanized antibody of claim 19, whereinthe multimerization domain comprises a human amino acid sequence. 21.The humanized antibody of claim 19, further comprising a linker locatedbetween the multimerization domain and the antibody.
 22. A humanizedantibody that inhibits human rhinovirus (HRV) infection of cellscomprising the amino acid sequence set forth in any of SEQ ID NO:1 and 3(HumA); SEQ ID NO:5 and 7 (HumB); SEQ ID NO:9 and 11 (HumC); SEQ IDNO:13 and 15 (HumD); SEQ ID NO:17 and 19 (HumE); SEQ ID NO:21 and 23(HumF); SEQ ID NO:25 and 27 (HumG); SEQ ID NO:29 and 31 (HumH); and SEQID NO:33 and 35 (HumI); or a subsequence thereof.
 23. The humanizedantibody of claim 22, wherein the antibody is an immunoglobulin moleculecomprising 2 full-length heavy chain polypeptides and 2 full-lengthlight chain polypeptides.
 24. The humanized antibody of claim 22,wherein the subsequence comprises a single chain, Fab, Fab′ or (Fab)₂fragment.
 25. The humanized antibody of claim 22, wherein the antibodyis linked with other identical or different antibodies to form amultimer.
 26. The humanized antibody of claim 25, wherein the multimercomprises a homo- or hetero-dimer, trimer, or tetramer.
 27. Thehumanized antibody of claim 25, wherein the different antibodies arehuman, humanized or non-human.
 28. A nucleic acid sequence encoding ahumanized antibody of claim 1 or 22 or a subsequence thereof.
 29. Anexpression cassette comprising the nucleic acid sequence of claim 28operably linked to an expression control element.
 30. A vectorcomprising the nucleic acid sequence of claim
 29. 31. The vector ofclaim 29, wherein the nucleic acid sequence is operably linked to anexpression control element.
 32. A cell comprising the nucleic acidsequence of claim
 28. 33. The cell of claim 31, wherein the cell isprokaryotic or eukaryotic.
 34. A pharmaceutical composition comprising ahumanized antibody of claim 1 or 5, and a pharmaceutically acceptablecarrier.
 35. The pharmaceutical composition of claim 34, wherein thecarrier is compatible with inhalation or nasal delivery to a subject.36. A method of inhibiting pathogen infection of a cell comprisingcontacting a pathogen or a cell with an amount of a humanized antibodyof claims 1 or 5, sufficient to inhibit pathogen infection of the cell.37. The method of claim 36, wherein the cell is present in a subject.38. The method of claim 37, wherein the cell is an epithelial cell. 39.The method of claim 37, wherein the cell expresses ICAM-1.
 40. A methodof inhibiting HRV infection of a cell comprising contacting HRV or acell susceptible to HRV infection with an amount of a humanized antibodyof claim 21 effective to inhibit HRV infection of the cell.
 41. Themethod of claim 40, wherein the cell is present in a subject.
 42. Themethod of claim 41, wherein the subject has or is at risk of havingasthma.
 43. The method of claim 40, wherein the antibody binds to anantigen present on the surface of the cell.
 44. The method of claim 40,wherein the cell expresses ICAM-1.
 45. The method of claim 40, whereinthe cell is an epithelial cell.
 46. The method of claim 40, wherein thehumanized antibody is administered locally.
 47. The method of claim 40,wherein the humanized antibody is administered via inhalation orintranasaly.
 48. A method of inhibiting HRV infection, inhibiting HRVprogression or treating HRV infection of a subject comprisingadministering to a subject having or at risk of having HRV infection anamount of a humanized antibody of claim 21 effective to inhibit, inhibitprogression or treat HRV infection of the subject.
 49. The method ofclaim 48, wherein the humanized antibody is administered locally. 50.The method of claim 48, wherein the humanized antibody is administeredvia inhalation or intranasaly.
 51. The method of claim 48, wherein thesubject has or is at risk of having asthma.
 52. The method of claim 48,wherein the subject is a newborn or between the ages of 1 to 5, 5 to 10or 10 to
 18. 53. A method of decreasing or inhibiting one or moresymptoms of the common cold in a subject comprising administering to asubject having a common cold an amount of a humanized antibody of claim21 effective to decrease or inhibit one or more symptoms of the commoncold in the subject.
 54. The method of claim 53, wherein the humanizedantibody is administered locally.
 55. The method of claim 53, whereinthe humanized antibody is administered via inhalation or intranasaly.56. The method of claim 53, wherein the subject has or is at risk ofhaving asthma.
 57. The method of claim 53, wherein the subject is anewborn or between the ages of 1 to 5, 5 to 10 or 10 to 18.